CN101063150B - Method for transducing bone marrow mesenchymal stem cells with human telomerase gene - Google Patents

Abstract

The present invention provides a method for human telomerase gene transduction bone marrow mesenchymal stem cells: the pGRN 145 plasmid is digested with Hpa I and Not I restriction endonucleases to obtain hTERT cDNA fragments, and using the same restriction endonucleases The pLXSN-hTERT recombinant expression vector was constructed by connecting the digested pLXSN vector with Dicer enzyme; after the pLXSN-hTERT recombinant expression vector suspension was prepared by the amphagocytic packaging cell line PA317 with LipofectAMINE liposome transduction kit, the human bone marrow mesenchymal Human bone marrow mesenchymal stem cells were transduced to construct human bone marrow mesenchymal stem cells highly expressing exogenous hTERT gene; human bone marrow mesenchymal stem cells were double screened with low concentration of hygromycin. The invention transduces human telomerase gene to human bone marrow mesenchymal stem cells, increases the expression level of this protein in cells, and achieves the purpose of improving the life cycle of bone marrow mesenchymal stem cells.

Description

Method for transducing bone marrow mesenchymal stem cells with human telomerase gene

technical field

The invention belongs to biological technology and relates to a new technology for constructing and screening bone marrow mesenchymal stem cells exogenously expressing human telomerase. This technique enables bone marrow mesenchymal stem cells to highly express exogenous human telomerase, thereby improving the life cycle of bone marrow mesenchymal stem cells.

Background technique

At present, the commonly used technologies that can be used to prolong the life cycle of cells and make cells immortal can be roughly classified into four types: Epstein-Barr virus (EBV) infection technology, simian virus 40 (SV40) large T antigen technology, and human papilloma type 16 Virus (HPV16) E6/E7 technology, telomerase gene technology. Co-cultivation of Epstein-Barr virus and cells to immortalize cells is the most widely used B lymphocytes. One of the most notable features of EB virus immortalized B lymphocytes is the increase in telomerase activity, which is the main reason for the immortalization of B lymphocytes. SV40 is a simple eukaryotic virus, and the T antigen fragment of SV40 is the most commonly used target fragment. When it is integrated into the nucleus of human target cells and expressed, it can lead to changes in cell proliferation and display a variety of tumor-related transformation phenotypes . But Shay et al. (1989) showed that, in most cases, the immortalization rate of SV40 transfected cells was very low. Papillomavirus E6 and hepatitis B virus (HBV) can enhance the proliferation ability of some special cells, but they are not commonly used viruses to make cells immortal. Human papillomavirus E6 protein activates telomerase through transcriptional regulation of the catalytic subunit of telomerase (hTERT); integration of the HBV enhancer upstream of the hTERT promoter can activate hTERT gene transcription in cis, and this cis-activation is Primary mechanisms of hTERT gene transcription and regulation of telomerase. Telomerase is a complex composed of RNA and protein. It is a special DNA polymerase that synthesizes DNA to the end of chromosomes. Once telomerase is activated, it will maintain the dynamic balance of telomere shortening and lengthening, so that cells can obtain the ability of unlimited proliferation and make them immortal. The results of various human immortalized cell experiments transformed with viruses demonstrate that telomerase activation may be a common step in cell immortalization. The most commonly used technology to prolong the life cycle of cells and make cells immortal is to construct an expression vector containing telomerase catalytic subunit (hTERT) and transfect cells by various methods.

Contents of the invention

The purpose of the present invention is to provide a method for transducing human telomerase gene into bone marrow mesenchymal stem cells, which is to transduce human telomerase gene into human bone marrow mesenchymal stem cells through transgenic technology, and improve the quality of human bone marrow mesenchymal stem cells. In order to improve the expression level of this protein, the purpose of improving the life cycle of bone marrow mesenchymal stem cells is achieved. Achieved through the following schemes:

(1) Construction of pLXSN-hTERT expression vector, pGRN145 plasmid (Geron Corporation, Menlo Park, CA) digested with Hpa I and Not I restriction endonucleases (Takara), separated and purified by agarose gel electrophoresis to obtain hTERT cDNA fragment, And connected with the pLXSN vector (Stratagene, La Jolla, CA) digested with the same restriction endonuclease to construct pLXSN-hTERT recombinant expression vector.

In the present invention, the pGRN 145 plasmid is digested with Hpa I and Not I restriction endonucleases, separated and purified by agar gel electrophoresis to obtain hTERT cDNA fragments, and the first step is to purify hTERT cDNA fragments.

In the present invention, the pLXSN carrier (Stratagene, La Jolla, CA) is digested with Hpa I and Not I restriction endonucleases, separated and purified by agar gel electrophoresis to obtain pLXSN carrier fragments, and the second step is to purify the expression vector fragments.

The human retrovirus vector used in the present invention adopts pLXSN vector to obtain pLXSN-hTERT recombinant expression vector for highly expressing hTERT.

(2) To construct human bone marrow mesenchymal stem cells highly expressing exogenous hTERT gene, according to the instruction manual of LipofectAMINE liposome transduction kit (Life Technologies, Grand Island, NY), through the amphagocytic packaging cell line PA317 (ATCC, Rockefeller, MA) after preparing the pLXSN-hTERT recombinant expression vector suspension, in 1,5-dimethyl-1,5-diazaundecylidene polymethyl bromide (Polybrene, Sigma, St.Louis, MO) Human bone marrow mesenchymal stem cells (Qiu Liyan et al., 2004) were transduced with pLXSN-hTERT expression vector for 8 hours with assistance. Thus, human bone marrow mesenchymal stem cells expressing exogenous hTERT gene highly efficiently were constructed.

(3) Using low concentrations (20 μg/ml and 50 μg/ml) of Hygromycin (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for double selection of human bone marrow mesenchymal stem cells transduced with exogenous hTERT gene A technique to improve the viability of bone marrow mesenchymal stem cells transduced with exogenous hTERT gene.

The beneficial effects of the present invention are:

(1) In the technology provided by the present invention, the pLXSN-hTERT expression vector that highly expresses hTERT connected with hTERT and pLXSN vector is used for transduction, so as to achieve the effect of constructing human bone marrow mesenchymal stem cells that highly express hTERT exogenous gene.

(2) Human bone marrow mesenchymal stem cells exogenously expressing hTERT constructed by the technology of the present invention were cultured in a low-solubility Hygromycin (Amresco, USA) double screening culture system, and human bone marrow mesenchymal stem cells not transduced with hTERT in the culture system were removed. mesenchymal stem cells, improving the success rate of constructing human bone marrow mesenchymal stem cells exogenously expressing hTERT.

Description of drawings

Figure 1: The expression vector pLXSN-hTERT that highly expresses hTERT.

Figure 2: Detection of hTERT gene expression by RT-PCR technology, Northern blotting technology and hTERT protein expression by Western blotting technology.

Figure 3: Analysis of surface antigens of hTERT-hMSCs at passage 95 by flow cytometry

Figure 4: Analysis of surface antigens of hTERT-hMSCs at passage 275 by flow cytometry

Figure 5: Analysis of surface antigens of normal 13th passage hMSCs by flow cytometry.

Figure 6: Karyotype analysis of cells by G-banding.

Figure 7: Life cycle analysis of immortalized bone marrow mesenchymal stem cells.

Figure 8: Analysis of the potential of bone marrow mesenchymal stem cell immortalized line to differentiate into fat, cartilage and osteoblasts.

Fig. 9: Results of colony formation assay in soft agar culture.

Figure 10: Results of tumorigenicity experiments in immunodeficient mice.

Detailed ways

The present invention will be further described in conjunction with specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

The technology of the present invention adopts Hpa I and Not I restriction endonucleases to cut pGRN145 plasmid, separates and purifies by agar gel electrophoresis to obtain hTERT cDNA fragment, and adopts the same restriction endonuclease digestion human retrovirus vector pLXSN vector The pLXSN-hTERT recombinant expression vector for highly expressing hTERT was constructed, as shown in Figure 1.

Specific steps are as follows:

1. Mass preparation of pGRN145 plasmid (alkaline lysis method):

1) Resuspend 500 ml of washed bacteria (containing pGRN145 plasmid) culture pellet in 18 ml of solution I (containing 50 mmol/L glucose, 25 mmol/L Tris·Cl (pH 8.0), 10 mmol/L liter EDTA (pH 8.0)).

2) Add 2 ml of freshly prepared lysozyme solution [10 mg/ml, dissolved in 10 mmol/L Tris·Cl (pH 8.0)] (When the pH of the solution is lower than 8.0, lysozyme cannot work effectively.)

3) Add 40 ml of newly prepared solution II [0.2 mol/L NaOH (dilute with 10 mol/L stock solution before use), 1% SDS], tightly cap the bottle, and slowly invert the number of centrifuged bottles times to thoroughly mix the contents. Leave at room temperature for 5-10 minutes.

4) Add 20 ml of ice-cooled solution III (60 ml of 5 mol/L potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of water). Seal the mouth of the bottle and shake the centrifuge bottle several times to mix the contents. At this time, two distinct liquid phases should no longer appear. Put it on ice for 10 minutes, a white flocculent precipitate should form.

5) Centrifuge at 4000 rpm for 15 minutes at 4°C to stop the rotor naturally. (If the bacterial fragments are not tightly attached to the wall, centrifuge again at 5000 rpm for 20 minutes, then transfer all the supernatant to another bottle as much as possible, and discard the viscous liquid remaining in the centrifuge tube. (Failure to form a dense precipitate The cause is usually due to insufficient mixing of Solution III with the bacterial lysate.)

6) Filter the supernatant into a 250ml centrifuge bottle with 4 layers of cheesecloth, add 0.6 volume of isopropanol, mix thoroughly, and place at room temperature for 10 minutes.

7) Centrifuge at 5000 rpm for 15 minutes at room temperature to recover nucleic acid (if centrifuged at 4°C, salt will also precipitate.)

8) Pour off the supernatant carefully, open the mouth of the bottle and invert the centrifuge bottle to drain the residual supernatant, and wash the precipitate and tube wall with 70% ethanol at room temperature. Pour off the ethanol and place the bottle upside down on a paper towel at room temperature to evaporate the last remaining traces of ethanol.

9) Dissolve the nucleic acid precipitate with 3 ml TE (pH 8.0).

10) Plasmid DNA was purified with polyethylene glycol.

Second, the preparation of hTERT cDNA fragments:

Using Hpa I, Not I digestion, the reaction system is as follows:

pGRN 145 plasmid 1 microliter

Hpa I 0.5 microliters

Not I 0.5 microliters

10X Buffer 1 microliter

Acetylated BSA 1 microliter

Deionized water 6 microliters

Total volume: 10 microliters

Incubate at 37°C for 16 hours, perform electrophoresis on 1% agarose, and observe under long-wave ultraviolet light until the inserted target band and carrier band are clearly separated, then use a scalpel to cut and recover the electrophoresis fragment, and use the gel recovery kit to recover the target band .

Three, the preparation of pLXSN vector:

The basic procedures for the large-scale preparation of pLXSN plasmids are the same as those for the large-scale preparation of pGRN145 plasmids, except that ampicillin is used instead of chloramphenicol as the screening antibiotic added to the liquid and solid LB medium. Using Hpa I, Not I digestion, the reaction system is as follows:

pLXSN plasmid 1 microliter

Hpa I 0.5 μL

Not I 0.5 microliters

10×Bufer 1 microliter

Acetylated BSA 1 microliter

6 microliters of deionized water

Total volume: 10 microliters

Incubate at 37°C for 16 hours, perform electrophoresis on 1% agarose, and observe under long-wave ultraviolet light until the inserted target band and carrier band are clearly separated, then use a scalpel to cut and recover the electrophoresis fragment, and use the gel recovery kit to recover the target band .

Fourth, the hTERT cDNA fragment is connected with the pLXSN vector to form the hTERT-pLXSN plasmid:

Vector and target fragment (hTERT) ligation reaction system:

pLXSN 1 µl

hTERT cDNA 1 microliter

T4 ligase Bufer(10x) 1 microliter

T4 ligase 0.5 μl

Deionized water 6.5 microliters

Total volume 10 microliters

The loading order of the above reaction is: first add the carrier, target fragment and deionized water into the reaction tube, bathe in water at 65°C for 5 minutes, immediately ice-bath to expose the sticky ends, and then add T4 ligase and ligase buffer. The ligation reaction was carried out at 16°C for 18 hours.

Five, prepare competent bacteria:

The following experiments were all done under sterile conditions. (1) Inoculate 0.1 ml of TOP10 strains into 6 ml of antibiotic-free LB liquid medium, shake and culture at 37°C at a speed of 200 r/min, and place the culture tube in an ice-water bath when the OD 600 value of the liquid reaches 0.4 . (2) In the ultra-clean workbench, aseptically dispense the bacterial solution, 1.5 ml per tube, centrifuge at 4000 rpm at 4°C for 5 minutes, and discard the supernatant. (3) Add 0.5 milliliters of ice-cold 0.1 mol/liter CaCl ₂ to each tube to suspend the bacteria. (4) Centrifuge at 4000 rpm at 4°C for 5 minutes, discard the supernatant, add 0.1 ml of ice-cooled 0.1 mol/L CaCl ₂ to resuspend the cells, and place in a 4°C refrigerator for use the next day.

6. Preparation of solid plate medium containing penicillin:

(1) Weigh 10 grams of tryptone, 5 grams of yeast extract, and 10 grams of NaCl, add 1000 ml of distilled water to fully dissolve them, and add 5 mol/L NaOH to adjust the pH to 7.0. (2) Take 10 250 ml Erlenmeyer flasks, add 1.5 grams of agar to each of them, and then add 100 ml of dissolved liquid LB medium. 1.034×10 ^5Pa autoclave for 20 minutes, store at room temperature for later use. (3) When preparing a solid LB plate, take out 100 ml of antibiotic-free solid LB, heat it in a microwave oven to melt it, put it at room temperature and wait for the temperature to drop to 50-55 ° C, add ampicillin (50 mg/ml) 0.3 ml, mix well and pour into a sterile plate, wait for it to solidify at room temperature and place it at 4°C.

7. Transform competent cells:

(1) Take a competent cell prepared the day before, add 5 microliters of the ligated plasmid product hTERT-pLXSN in the third step, mix well, and then ice-bath for 30 minutes. (2) Water bath at 42°C for 90 seconds, and ice bath for 2 minutes immediately after heat shock. (3) Add 400 microliters of liquid LB medium without antibiotics, and incubate at 37° C. for 1 hour. (4) Centrifuge at 4000rpm for 10 minutes, discard 400 microliters of the supernatant, take the remaining 100 microliters of resuspended transformed bacteria, spread them all on LB solid plates containing antibiotics, and incubate at 37°C for 16 hours, and the transformed colonies can be seen growing.

8. Small-scale cultivation, amplification and plasmid extraction of transformed bacteria:

A small amount of plasmid extraction kit from v-gene company was used. (1) Take a single transformed colony on the LB solid plate containing ampicillin, inoculate it into 6 ml of LB liquid medium containing ampicillin, shake it at 37°C for 16 hours at a speed of 200 r/min, and take the bacterial liquid 1.5 milliliters were placed in a centrifuge tube, centrifuged at 4000rpm for 5 minutes, and the supernatant was discarded. (2) Add 250 microliters of solution I to fully suspend the bacteria. (3) Add 250 microliters of solution II, pay attention to mixing, at this time the liquid becomes clear and viscous, and let it stand at room temperature for 2 minutes. (4) Add 400 microliters of solution III and let stand at room temperature for 2 minutes. (5) Centrifuge at 12000 rpm for 10 minutes, and recover the supernatant. (6) Put the supernatant in a plasmid recovery column and centrifuge at 5500 rpm for 1 minute. (7) Discard the waste liquid, add 500 microliters of washing buffer, and centrifuge at 5500 rpm for 1 minute. (8) Repeat step 7 once. (9) Centrifuge at 12,000 rpm for 1 minute, add 60 microliters of TE buffer and leave at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute, and store the resulting plasmid solution at -20°C for use.

Nine, the screening of recombinant bacteria:

Take a single transformed colony on the solid plate, inoculate it into 0.1 ml of LB liquid medium containing ampicillin, shake it at 37°C for 16 hours at a speed of 200 r/min, and then screen the target plasmid by PCR. Human telomerase gene upstream primer sequence: 5'-CTC TCC CCC TTG AAC CTCCTC GTTC C; downstream primer sequence: 5'-AGG ACA CCT GGC GGA AGG AG.

Specific reaction system:

Upstream primer sequence (10 micromol/L) 1 microliter

Downstream primer sequence (10 micromol/L) 1 microliter

MgCl (25 mmol/L) 1.2 microliters

Bacteria solution 0.5 microliters

dNTP (4 mmol/L) 1 microliter

Taq DNA polymerase (10 units/μl) 0.4 μl

Buffer(10x) 2 microliters

Deionized water 12.9 microliters

Total volume 20 microliters

Operation process:

First, in a sterile 0.2 ml centrifuge tube, add various reaction substances except Taq polymerase in turn, place in a 94°C water bath for pre-denaturation for 5 minutes, and immediately place it in an ice water bath. Add Taq polymerase followed by PCR cycle. The conditions are as follows: denaturation at 94°C for 30 seconds; annealing at 52°C for 30 seconds; extension at 72°C for 80 seconds; 25 cycles. After the end of the PCR cycle, extend at 72°C for 7 minutes. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, observed under a portable ultraviolet analyzer, and positive clones were obtained. The plasmids of positive clones were extracted by alkaline lysis.

10. Identification of pLXSN-hTERT recombinant plasmid

Using restriction map method, use Hpa I; Not I for enzyme digestion.

Hpa I; Not I enzyme digestion system of pLXSN-hTERT:

pLXSN-hTERT 1 µl

Hpa I 0.5 microliters

Not I 0.5 microliters

10X Bufer 1 microliter

Acetylated BSA 1 microliter

Deionized water 6 microliters

Total volume: 10 microliters

The digestion reaction of the above reaction system was incubated at 37° C. for 2 hours, after 1% agarose gel electrophoresis, stained with ethidium bromide, observed under a short-wave ultraviolet analyzer and photographed under a projected ultraviolet analyzer.

Example 2

The technology of the present invention adopts the LipofectAMINE liposome transduction kit, and carries out the preparation of the recombinant expression carrier suspension and the transduction of human bone marrow mesenchymal stem cells according to the usage method of the kit. According to the instructions of the kit, the amphagocytic packaging cell line PA317 was used to prepare the recombinant virus vector suspension, and 200 μg/ml hygromycin was used to screen the recombinant expression vector for 4 days. With the aid of 8 μg/mL of 1,5-dimethyl-1,5-diazaundecamethylene polymethyl bromide (Polybrene; Sigma, St. Louis, MO), in a 10-cm Petri dish 2.0×10 ⁵ human bone marrow mesenchymal stem cells were transduced with recombinant expression vectors for 8 hours in (Maxisorp, Nunc, Denmark). Cells were then washed with phosphate-buffered saline (PBS), and cells were first selected with 20 μg/ml hygromycin for 7 days. After 7 days, the cells were washed with normal culture medium, and the cells were selected for the second time with 50 μg/ml hygromycin for 7 days. Afterwards, subculture with normal complete medium.

Example 3

The technology of the present invention adopts RT-PCR technology and Northern blotting technology to detect the expression level of hTERT gene in transduced human bone marrow mesenchymal stem cells. The results showed that the bone marrow mesenchymal stem cells transduced with the hTERT gene effectively expressed the hTERT gene, see Figure 2A, 2B, the figure shows (A) RT-PCR was used to detect the expression of the hTERT gene, including the Marker (3kb) swimming lane, Lane 1: expression of hTERT gene and β-actin gene in hTERT-hMSCs at passage 95, lane 2: expression of hTERT gene and β-actin in hTERT-hMSCs at passage 275, lane 3: normal hMSCs at passage 13 Expression of hTERT gene and β-actin gene in cells, lane 4: RT-PCR with water as control. (B) Detection of hTERT gene expression by Northern blotting, including lane 1: expression of hTERT gene and β-actin gene in 95th passage hTERT-hMSCs cells, lane 2: hTERT gene and β-actin gene in 275th passage hTERT-hMSCs cells -Expression of actin gene, lane 3: expression of hTERT gene and β-actin gene in normal 13th passage hMSCs cells.

1. RT-PCR technology detection:

The primer sequence for RT-PCR detection of exogenous human telomerase gene was designed (upstream: 5'-CTC TCCCCC TTG AAC CTC CTC GTTC; downstream: 5'-AGG ACA CCT GGC GGA AGGAG), and the product fragment was 408bp. The primer sequence of the control β-actin (upstream: 5'-CAT CTC TTG CTCGAA GTC CA-3'; downstream: 5'-ATC ATG TTT GAG ACC TTC AAC A), the product fragment is 318bp. RT-PCR amplification was carried out, and the expression of the exogenous telomerase gene mRNA was observed and detected after amplified products were subjected to agarose gel electrophoresis.

2. RNA extraction steps: (Using TRIZOL total RNA extraction kit from Invitrogen Company, according to the instruction manual)

(1) Add 1 ml of Trizol (Invitrogen, Beijing, China) to a culture flask bottom area of 10 cm ² for homogenization.

(2) Centrifuge at 12000 g for 10 minutes at 4° C., and transfer the supernatant to a clean RNase-free centrifuge tube (RNA exists in the supernatant).

(3) Leave it at room temperature for 5 minutes. Add 0.2 ml of chloroform to every 1 ml of Trizol, cover the test tube, shake vigorously for 15 seconds, incubate at room temperature for 3 minutes, and centrifuge at 12,000 g at 4°C for 15 minutes. After centrifugation, the mixture was divided into three layers, the red phenol-chloroform layer at the bottom, the middle phase and the colorless upper aqueous phase, and RNA only existed in the upper aqueous phase.

(4) Transfer the aqueous phase to a clean test tube, add 0.5 ml of isopropanol to wash the precipitate, and centrifuge at 12,000 g for 5 minutes at 4°C.

(5) Slightly dry the RNA precipitation (air drying for 5-10 minutes), dissolve the RNA with an appropriate amount of deionized water, and detect the quality of the RNA by electrophoresis (the RNA cannot be completely dried, otherwise it will be difficult to dissolve).

3. Steps of reverse transcription (according to the instructions of the reverse transcriptase kit):

1) cDNA synthesis reaction system:

(1) Mix the primers, RNA and four deoxyribonucleic acid mixtures (dNTP Mix) in a 0.2 ml centrifuge tube, and adjust the volume to 11 microliters with diethylpyrocarbonate (DEPC)-treated water.

Ingredients Quantity

50μM Oligo(dT)18 primer 0.5μg

Total RNA 0.1-5 μg

Deionized water (nuclease-free) to 11 μl

(2) The above mixture was incubated at 70°C for 5 minutes, and then immediately placed on ice.

(3) Shake the 5× cDNA synthesis buffer for 5 seconds before use.

(4) Add the following ingredients to the centrifuge tube:

Ingredients Quantity

5× reaction buffer 4 μl

10mM dNTP Mix 2 microliters

Ribonuclease inhibitor 20 units

Deionized water (nuclease-free) to 19 μl

After incubation at 37°C for 5 minutes, 200 units of reverse transcriptase were added. After briefly centrifuging and mixing the above mixture, immediately place it at 42°C for 60 minutes to synthesize cDNA.

Immediately after the reaction is completed, place at 70°C for 10 minutes to inactivate the reverse transcriptase.

The synthesized first-strand cDNA was stored at -20°C for future use.

2) PCR amplification of exogenous telomerase gene cDNA:

PCR reaction system: (total volume 50 microliters)

Reaction Component A Reaction

10× PCR buffer 5 microliters

2 millimolar dNTP Mix 5 microliters

Primer 1 (10 micromole) 1 microliter

Primer 2 (10 micromole) 1 microliter

Taq DNA Polymerase 1 microliter (5 units in total)

Template DNA 2 microliters

25mmol MgSO4 2 microliters

Deionized water 33 microliters

Final volume 50 microliters

Conditions of PCR amplification reaction: pre-denaturation at 94°C for 120 seconds, then 94°C for 30 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds, a total of 30 cycles, take 15 microliters of PCR The product was electrophoresed on a 2% agarose gel to detect the amplification result.

3) Northern-blotting technology detection:

RNA electrophoresis:

a. Prepare 1% agarose formaldehyde denaturing gel:

Weigh 3 g of agarose, add 30 ml of 10×MOPS (3-[N-morpholine]-propanesulfonic acid), 255 ml of 0.1% DEPC-H ₂ O and heat until the agarose is completely melted, then place it at room temperature and cool to 60 ℃, add 16.2 ml of 37% formaldehyde solution, mix well and make gel.

b. Loading and electrophoresis:

Take 20 micrograms of RNA samples, add 20 microliters of RNA loading buffer, mix well, denature in a 65°C water bath for 5 minutes, and put the gel into an electrophoresis tank filled with 1×MOPS electrophoresis buffer, pre-electrophoresis for 10 minutes, and then Add the sample, electrophoresis at 100 volts until the sample runs out of the sample hole, and then adjust the voltage to 60 volts for electrophoresis until the bromophenol blue dye migrates to the downstream 3/4 of the gel. After electrophoresis, use 0.25 μg/ml ethidium bromide After EB staining, the gel was transferred to a UV image analyzer for analysis.

c. Transfer film:

Gels containing formaldehyde must be rinsed several times with DEPC water to remove formaldehyde; cut off the upper left corner of the gel as a mark. Transfer the RNA on the gel to the nylon membrane by siphoning through the pores of a paper towel. The transfer time was 16 hours, and then the nylon membrane was vacuum-dried at 80° C. for 2 hours, and then stored at room temperature.

d. Label the probe:

Take 25 nanograms of probes, label with random primer labeling kit and ^[32P] dCTP after denaturation, store at -20°C, and denature again before use.

e. Hybridization and autoradiography

1) Pre-hybridization and hybridization: put the membrane in a medium containing 6×citric acid buffer (SSC), 5×Denhardt buffer, 0.5% sodium dodecyl sulfate (SDS) and 100 μg/ml denatured salmon sperm DNA. In the prehybridization solution, prehybridize at 58°C for 1 hour. Pour out the pre-hybridization solution, replace it with a hybridization solution containing 6×SSC, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA and 50 μl of isotope-labeled probe, and hybridize at 58° C. for 16 hours.

2) Membrane washing and autoradiography: wash the nylon membrane in 1×SSC, 0.1% SDS solution at room temperature for 20 minutes; wash the membrane with 0.2×SSC, 0.1% SDS at 68° C. for 3 times, 20 minutes each time. After the film was naturally dried at room temperature, it was wrapped with plastic wrap, and autoradiography was performed with X-ray film in the dark room. After exposure at -70` for 7 days, conventional development and fixing were performed.

Example 4

The present invention adopts Western blotting technology to detect the expression of hTERT protein in transduced human bone marrow mesenchymal stem cells. The results showed that bone marrow mesenchymal stem cells transduced with hTERT gene effectively expressed hTERT protein, see Figure 2C, in the figure (C) Western blotting was used to detect the expression of hTERT protein, including lane 1: hTERT-hMSCs cells at passage 95 Expression of hTERT protein and β-actin protein, lane 2: expression of hTERT protein and β-actin protein in hTERT-hMSCs at passage 275, lane 3: expression of hTERT protein and β-actin protein in normal hMSCs at passage 13 .

a. Protein sample preparation:

The culture medium in the bone marrow mesenchymal stem cells and the transgenic bone marrow mesenchymal stem cells grown adherently to 90% confluence was removed. Add 4 ml of 4°C pre-cooled PBS buffer to wash the cells 3 times, discard the PBS buffer and place the culture flask on ice;

Add 10 ml of phenylmethylsulfonyl fluoride (PMSF) (100 mmol/L) to 1 ml of cell lysate (Roche, New Jersey, USA), shake well and place on ice. Add 400ml of PMSF-containing lysate to each bottle of cells, lyse on ice for 30 minutes, transfer the cell debris and lysate to a 1.5ml centrifuge tube; centrifuge at 12,000 rpm for 5 minutes at 4°C. Aliquot the centrifuged supernatant to a 0.5 ml centrifuge tube and store at -20°C for later use.

b. Determination of protein content (see Table 1)

To make a standard curve: take out 1 mg/ml BSA from -20°C, melt it at room temperature, and set it aside. Take 18 1.5ml centrifuge tubes, in a group of 3, marked as 0mg, 2.5mg, 5.0mg, 10.0mg, 20.0mg, 40.0mg respectively. Add various reagents to each tube according to the table below. After mixing, let stand at room temperature for 2 minutes. Colorimetric analysis on a biospectrophotometer.

Table 1 Determination of protein content

0 micrograms 2.5 micrograms 5.0 micrograms 10.0 micrograms 20.0 micrograms 40.0 micrograms 1 mg/ml BSA - 2.5 microliters 5.0 microliters 10.0 microliters 20.0 microliters 40.0 microliters 0.15 mol/L NaCl 100 microliters 97.5 microliters 95.0 microliters 90.0 microliters 80.0 microliters 60.0 microliters G250 Coomassie brilliant blue solution 1ml 1ml 1ml 1ml 1 ml 1ml

Detection of sample protein content: take a sufficient amount of 1.5 ml centrifuge tubes, and add 1 ml of Coomassie Brilliant Blue solution stored at 4°C to each tube. After 30 minutes at room temperature, it was used to measure protein. Take a tube of Coomassie Brilliant Blue plus 0.15 mol/L NaCl solution 100ml, mix it and leave it for 2 minutes as a blank sample, pour the blank into a cuvette and measure the blank sample under the procedure of making a standard curve. Discard the blank sample, wash the cuvette twice with absolute ethanol, and wash once with sterile water. Take a tube of Coomassie Brilliant Blue, add 95 ml of 0.15 mol/L NaCl solution and 5 ml of the protein sample to be tested, mix well and let stand for 2 minutes to test the sample.

c. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

After installing the gel device, fill the gel with 8% separating gel, add a layer of water, wait for the gel after the liquid seal to gel, pour off the water on the upper layer of the gel and dry the water with absorbent paper; pour 5% concentrated gel, put Insert the comb into the stacking gel. After the stacking gel is solidified, pull out the comb; cook the sample for 5 minutes before loading to denature the protein, add 5×SDS loading buffer, load 50 micrograms of protein sample; electrophoresis at 60 volts After 3-4 hours, observe the position of bromophenol blue to stop the electrophoresis and transfer to the membrane.

d. Protein transfer

The treatment of nitrocellulose membrane (Immobilon-NC membrane, Millipore, USA): immerse in methanol for 15 seconds, then wash with sterilized water for 2 minutes, and finally transfer to transfer buffer to equilibrate for 5 minutes;

Gel treatment: wash the gel with sterilized water, then soak in transfer buffer for 15 minutes;

Film transfer: Assemble the transfer device, and transfer the film at 60 volts for 2 hours under cooling conditions;

Check the transfer effect: After transfer, stain with 1×Ponceau staining solution for 5 minutes to check the protein on the membrane. Allow the film to dry for later use.

e. Immune response

After soaking the membrane with Tris salt buffer solution (TBS) from bottom to top, move it to a plate containing blocking solution, and block for 1 hour;

Dilute the primary antibody with Tween-Tris salt buffer (TBST) to an appropriate concentration (dissolve in TBST at 1:20,000), incubate at room temperature for 3-4 hours, or overnight at 4°C; then decolorize with TBST on a shaker at room temperature Rinse twice, 10 minutes each time; then rinse with TBS for 10 minutes;

Prepare the secondary antibody dilution (dissolved in TBST at 1:5,000) and contact with the membrane, incubate at room temperature for 2 hours, then rinse twice with TBST on a decolorizing shaker at room temperature, each time for 10 minutes; then rinse once with TBS , for 10 minutes, to carry out the chemiluminescent reaction.

f. Chemiluminescent color reaction

The membrane after antibody reaction was transferred to TSM solution (0.1mol/l Tris-HCl pH8.0, 0.1mol/LNaCl, 0.01mol/L MgCl ₂ ), 5 minutes×2; then add the chromogenic substrate 5-bromo-4 - Chloro-3-indophosphoric acid/nitroblue tetrazolium (BCIP/NBT), develop color at room temperature in the dark; TE buffer (10mM Tris-HCl, 1mM EDTAPH8.0) terminates the reaction.

Example 5

The invention adopts a flow cytometry method to detect the surface markers of the transduced human bone marrow mesenchymal stem cells. The results showed that the surface markers of bone marrow mesenchymal stem cells transduced with hTERT gene were consistent with those of normal human bone marrow mesenchymal stem cells. The surface antigens of hTERT-hMSCs cells at passage 95 are shown in Figure 3, the surface antigens of hTERT-hMSCs cells at passage 275 are shown in Figure 4, and the surface antigens of normal hTERT-hMSCs cells at passage 13 are shown in Figure 5. Their cell surface antigens all showed CD29 (Panel D), CD44 (Panel A), CD105 (Panel B) and CD166 (Panel C) were positive (+), CD34 (Panel H), CD45 (Panel G), CD117 (Panel Panel E) and HLA-DR (panel F) were negative (-).

Detection process:

1. When hTERT-hMSCs of different passages grow to about 60-80% confluent, trypsin digestion solution (Trysin-EDTA digestion solution, Jinuo, Hangzhou, China) digests the cells, and washes the cells 3 times with PBS buffer;

2. The collected cells were resuspended in PBS buffer, and the cell mass was filtered through a 400-mesh nylon sieve to collect well-dispersed single cell populations;

3. Add various fluorescein-labeled monoclonal antibodies, mix the cell suspension, and incubate at 4°C in the dark for 30 minutes;

4. Wash the cell solution twice with ethylenediaminetetraacetic acid-bovine serum albumin-phosphate buffer solution (EDTA-BSA-PBS, Shanghai Sangon, Shanghai, China), centrifuge the cell suspension at 300×g for 5 minutes, wash Remove unbound mAb. Cells were resuspended in EDTA-BSA-PBS buffer;

5. Resuspend the cell population with EDTA-BSA-PBS buffer, filter the cells with a 400-mesh nylon mesh, adjust the cell density to 2×10 ⁶ cells/ml, the total volume is 1 ml, and 500 μl is used for flow analysis. 500 microliters spare;

6. The experimental design is as follows: unlabeled cell population (blank control), mouse IgG (isotype antibody), single-stained samples of various cell populations (experimental group), FITC-mAb (fluorescence compensation 1), PE - mAb (fluorescence compensation 2);

7. Loading blank control and mouse IgG isotype antibody to adjust the voltage of fluorescence detector (FL2);

8. Use PE-mAb and FITC-mAb to adjust the fluorescence compensation of FL2;

9. Finally, load each cell group that is single-stained, and set a gate to delineate the cell group to analyze the flow cytometry results.

Example 6

The present invention uses the Giemsa staining method [2] to analyze the karyotype of transduced human bone marrow mesenchymal stem cells. The results showed that the karyotype of bone marrow mesenchymal stem cells transduced with hTERT gene was consistent with that of normal human bone marrow mesenchymal stem cells, see Figure 6, the karyotype of cells was analyzed by G-banding: (A) 95th passage hTERT -The karyotype of hMSCs cells, (B) the karyotype of the 275th passage hTERT-hMSCs cells, (C) the karyotype of the normal 13th passage hMSCs cells. Their cell chromosome length, centromere position, secondary constriction, presence or absence of satellite, arm ratio, centromere index and relative length indicated that the karyotype of hTERT-hMSCs cells was consistent with that of normal hMSCs cells. , transformation and other contamination did not occur during passage.

Detection process:

1.35mm petri dish was routinely cultured for 48 hours (exponential growth phase), replaced with 2 ml of fresh culture medium, added colchicine (prepared working solution 10 μg/ml) to a final concentration of 0.2 μg/ml, mixed gently, and routinely cultured for 4 Hour.

2. Digest the cells with trypsin, collect them into a centrifuge tube, rinse the culture dish with normal saline several times, and confirm that all the cells are collected into the tube.

3. Centrifuge at 1000 rpm for 10 minutes.

4. Discard the supernatant, add 0.5 ml of hypotonic solution (prepared with 0.4% potassium chloride: 0.4% sodium citrate 1:1) dropwise into the centrifuge tube, flick evenly, then make up 1 ml, mix well, and place in a water bath at 37°C. Infiltrate for 5 minutes. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.

5. Add 2 ml of fixative solution (methanol:glacial acetic acid 3:1), and centrifuge at 1000 rpm for 3 minutes.

6. Discard the supernatant, add 4 ml of fixative solution, blow evenly, cover and fix at room temperature for 40 minutes. Centrifuge at 1000 rpm for 3 minutes.

7. Discard the supernatant, add 2 ml of fixative, blow evenly, cover and fix at room temperature for 20 minutes. Centrifuge at 1000 rpm for 3 minutes.

8. Remove the supernatant, leave about 0.3 ml of the mixed solution, beat well, drop the tablet, drop at a height of 50-100 cm. (Slides should be rinsed according to the routine of laboratory glass items, and set aside in a water bath in a refrigerator at 4°C)

9. Put the slide in a 65°C oven overnight

10. Take the slides out of the oven and stain with Giemsa for 10 minutes.

11. Rinse with tap water and air dry naturally.

Example 7

The present invention adopts the method of cell subculture to detect the life cycle of transduced human bone marrow mesenchymal stem cells, and the result shows that the bone marrow mesenchymal stem cells transduced with hTERT gene still have vigorous vitality after cultured for 290 generations. The primary hMSC can only be passaged for 25 passages, see Figure 7, the life cycle analysis of hTERT-transduced bone marrow mesenchymal stem cells. hTERT-hMSCs were cultured for 290 passages (indicated by rectangle boxes), and primary hMSCs were cultured for 25 passages (indicated by dots).

Passage process:

1. After the primary cultured cells or bone marrow mesenchymal stem cells transduced with hTERT gene cover 90% of the bottom area of the culture flask, the cells are subcultured;

2. Suck out the culture medium in the culture flask, wash the cells 2-3 times with PBS buffer, add 2 mL of Trypsin-EDTA digestion solution, gently shake the culture flask back and forth, and digest the cells at room temperature for 3 minutes;

3. Observe the morphological changes of the cells under an inverted microscope. When the cells retract from the spindle shape, add 2mL MEM-α complete culture medium (Gibco BRL, USA) to terminate the digestion reaction;

4. Gently blow the cells pasted on the bottom with a pipette until all the cells are suspended in the culture medium;

5. Collect the cells by centrifugation at 1,000 rpm for 5 minutes, and resuspend them in MEM-α complete culture medium;

6. Count the total number of cells, inoculate and subculture at a ratio of 1:3, and record the cells as P1 generation;

7. When the cells are 90% confluent, continue to subculture at a ratio of 1:3, replace the same volume of fresh MEM-α complete culture medium every 3 days, and record it as P2 generation, P3 generation, P4 generation,... …

Example 8

The invention adopts the method of fat induction liquid induction to analyze the potential of transduced human bone marrow mesenchymal stem cells to differentiate into fat cells. The results show that bone marrow mesenchymal stem cells transduced with hTERT gene can differentiate into adipocytes under the induction of fat inducing solution, see Figure 8A, 8B, 8C, which is the immortal line of bone marrow mesenchymal stem cells to fat, cartilage, and osteogenesis Analysis of cell differentiation potential. The 95th passage (A) and 275th passage (B) cells of immortalized bone marrow mesenchymal stem cells were induced by fat-inducing solution for 14 days, and stained with oil red O, the cells with orange-red lipid droplet deposition were seen under the inverted phase-contrast microscope, and the cells in the control group were not Only the nuclei of induced cells were stained blue by hematoxylin (C).

Induction process:

The 25th and 65th passage hTERT-hMSCs cells were taken, digested and made into a single cell suspension, and inoculated on a 35 mm diameter petri dish at 2×10 ⁴ per dish, a total of 12. After the cells reached 80-90% confluence, add adipocyte induction medium (α-MEM culture medium, 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 10 ng /ml insulin-like growth factor-I (IGF-I), 1 micromol dexamethasone, 0.5 mmol 1-methyl-3-isobutyl-xanthine, 100 micromol indomethacin), control group 6 Plates with conventional culture medium. Change the medium every 3-4 days, and stain with Oil Red O after 3 weeks.

Oil Red O staining of induced cells

After 21 days, 6 dishes from the experimental group and the control group were taken for Oil Red O staining. After discarding the culture medium, the culture dish was washed 3 times with PBS, fixed with 10% formaldehyde at room temperature for 10 minutes, incubated with saturated Oil Red O dye solution for 2-3 hours, washed with 60% isopropanol to remove excess dye solution, and washed with distilled water for 2 minutes. Mayer's hematoxylin solution was stained for 15-30 minutes, differentiated with 1% hydrochloric acid (prepared with 70% ethanol) for 2 seconds, washed with distilled water for several minutes, blued with alkali alcohol, mounted with neutral gum, and observed.

Example 9

The invention adopts the method of induction of chondrogenic induction liquid to analyze the potential of transduced human bone marrow mesenchymal stem cells to differentiate into chondrogenic cells. The results show that bone marrow mesenchymal stem cells transduced with hTERT gene can differentiate into chondrogenic cells under the induction of chondrogenic induction medium, see Figure 8D, 8E, 8F, 8G, 8H, 8I, bone marrow mesenchymal stem cell immortalized line After being induced by chondrogenic medium for 28 days, Alcian blue staining showed blue granules in the cytoplasm and extracellular matrix of the 95th passage (D) and 275th passage (E), while the uninduced cells in the control group were only stained with red nuclei (F). The expression of type II collagen was detected by immunohistochemistry. It can be seen that the cytoplasm and intracellular matrix of the 95th generation (G) and 275th generation (H) induction groups were stained red, while the control group (I) only the nucleus was stained blue.

Induction process:

Using the high-density drop culture method, transfer the passaged cell suspension to a 15-ml polypropylene centrifuge tube at a density of 2.5×10 ⁶ /0.5 microliter medium, and centrifuge at 500 rpm for 5 minutes. Make it aggregate in the cone The bottom of the shaped centrifuge tube was formed into a 'droplet' shape for induction, and each experimental tube was added into a cartilage induction culture solution, and the solution was changed every other day, 4/5 of the total amount of the solution was changed each time.

Alcian blue staining of chondrocytes

After termination of induction, wash with PBS (pH 7.2) 3 times for 1 minute each time, fix the specimen with 4% paraformaldehyde, rinse with distilled water, stain with Alcian blue 8GX (Amresco USA) for 30 minutes, rinse with 3% acetic acid and distilled water. Nuclei were stained for 4 minutes with eukaryotic solution. Seal the slides with resin and observe under the microscope.

Detection of Type II Collagen Expression by Immunohistochemical Method

According to the EnVision two-step method [3] immunohistochemical operation steps. The primary antibody is mouse anti-human type II collagen monoclonal antibody, the secondary antibody is horseradish peroxidase-labeled goat anti-mouse secondary antibody, 3-amino-9-ethylcarbazole (AEC) color development, neutral gum cover film.

Example 10

The present invention adopts an induction method of osteogenic induction liquid to analyze the potential of transduced human bone marrow mesenchymal stem cells to differentiate into osteoblasts. The results show that bone marrow mesenchymal stem cells transduced with hTERT gene can differentiate into osteoblasts under the induction of osteogenic induction medium, see Figure 8J, 8K, 8L, 8M, 8N, 8O, bone marrow mesenchymal stem cell immortalized line Von Kossa staining showed calcium phosphate deposition after being induced with osteogenic induction solution for 28 days. It can be seen that the calcium phosphate deposition sites in the cytoplasm and intracellular matrix of the 95th passage (J) and 275th passage (K) induced groups were stained black. While the control group (L) was not stained black. NBT/BCIP method showed alkaline phosphatase activity. It can be seen that the cytoplasm and intracellular matrix of the 95th passage (M) and 275th passage (N) induced groups were stained dark blue. In the control group (O), only the nuclei were stained light blue. Scale bar: 100 microns.

Induction process:

Digest the 25th and 65th passage hTERT-hMSCs into a single cell suspension, inoculate in cell flasks at a density of 3×10 ⁴ /ml, a total of 12 flasks, after the cells reach 80%-90% confluence 6 bottles of the experimental group were added containing 0.1 micromole dexamethasone, 50 micrograms/ml ascorbic acid, 10 millimolar β-glycerol phosphate culture solution (10% FBS) for induction, and 6 bottles of the control group were cultivated with conventional culture solution. The medium was changed every three days and observed under an inverted microscope.

von kossa stain

After termination of induction, wash with PBS (pH 7.2) 3 times, 1 minute each time, fix the specimen with 4% paraformaldehyde, incubate with 5% sodium thiosulfate for 30 minutes, rinse with distilled water, and then stain with 1% silver nitrate solution under ultraviolet light After 30 minutes, the black matter floating on the cell surface was washed away with distilled water, and stained with 5% sodium thiosulfate for 2 minutes, rinsed with distilled water and observed.

alkaline phosphatase staining

After termination of induction, wash with PBS (pH 7.2) 3 times, 1 minute each time, fix with 1% paraformaldehyde containing 7.5% sucrose at room temperature for 10-20 minutes, and then stain with alkaline phosphatase kit. According to the manufacturer’s instructions, equilibrate with substrate buffer first, and stain in freshly prepared 5-bromo-4-chloro-3-indophosphoric acid/nitroblue tetrazolium (BCIP/NBT) staining solution for 30 minutes at room temperature and dark Above, wash with water. Observe differentiated cells under a 100X inverted microscope.

Example 11

The invention adopts the method of soft agar colony formation test to analyze the tumorigenicity of transduced human bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells transduced with hTERT gene have no tumorigenicity, see Figure 9, in the figure A: normal bone marrow mesenchymal stem cells at the 13th passage culture on the first day, B: normal bone marrow mesenchymal stem cells at the 13th passage Day 4 of subculture, C: normal bone marrow mesenchymal stem cells at passage 13, day 10 of culture, D: immortalized line of bone marrow mesenchymal stem cells at passage 95, day 1 of culture, E: immortal line of bone marrow mesenchymal stem cells at passage 95 The 4th day of subculture, F: the 95th passage of immortalized bone marrow mesenchymal stem cell culture, the 10th day of culture, G: the 10th day of culture of the 275th passage of immortalized bone marrow mesenchymal stem cell line, H: the 1st day of culture of the 275th passage of immortalized bone marrow mesenchymal stem cell line The 4th day of culture at passage 275, I: the 10th day of culture at passage 275 of immortalized bone marrow mesenchymal stem cells, J: the first day of NIH-3T3 cell culture, K: the fourth day of NIH-3T3 cell culture, L: the NIH- The 10th day of 3T3 cell culture. Scale bar: 100 microns.

Soft agar colony formation test procedure:

(1) Take the cells in the logarithmic growth phase, digest them with 0.25% trypsin and blow gently to make them into single cells, count the viable cells, and adjust the cell density to 1× with basic cell culture medium containing 20% fetal bovine serum 10 ⁶ cells/liter. Then make gradient multiple dilutions according to the experimental requirements.

(2) Two low-melting-point agarose solutions of 1.2% and 0.7% were prepared with distilled water, and kept at 40° C. without solidification after autoclaving.

(3) After mixing 1.2% agarose and 2× complete medium (containing 2× antibiotics and 20% calf serum) at a ratio of 1:1, take 3 ml of the mixture and inject it into a 6 cm diameter dish, cool and solidify , can be used as the bottom agar and placed in a CO ₂ incubator for later use.

(4) After mixing 0.7% agarose and 2× complete medium in a sterile test tube at a ratio of 1:1, add 0.2 ml of cell suspension to the tube, mix well, and inject 1.2% In the agarose bottom plate, double agar layers are formed gradually. After the upper layer of agar is solidified, place it in a 5% CO ₂ incubator at 37°C for 10-14 days.

(5) Place the plate under an inverted microscope to observe the number of cell clones. Calculate formation rate.

Example 12

The invention adopts the method of immunodeficiency mice to analyze the tumorigenicity of transduced human bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells transduced with hTERT gene have no tumorigenicity, see Figure 10, in the figure A: side subcutaneous injection of immortalized bone marrow mesenchymal stem cells (photographed after six months), B: side subcutaneous injection Inject the positive cells of the human cervical cancer cell line Hela cells (photographed after 60 days), subcutaneously inject the bone marrow mesenchymal stem cell immortalized line in the side of the immunodeficient mice, and the positive cells of the human cervical cancer cell line Hela cells, after two weeks Observe for tumor formation. The tumor formation rate of positive cells in the control group was 15/15, while the mice injected subcutaneously with the immortal line of bone marrow mesenchymal stem cells were observed for six months. No tumor formation was found.

Basic steps: (1) Take the cells in the logarithmic growth phase, digest them with 0.25% trypsin and pipette gently to make them single cells, neutralize the trypsin with serum-containing medium, and centrifuge and wash twice with serum-free medium , suspend the cells in PBS, count the viable cells, adjust the cell concentration to 1×10 ⁷ cells/ml, and set aside. Non-obese diabetic/severe combined immunodeficiency mice, weighing 20-22 grams, 4-6 weeks old, use a syringe to draw 0.3 ml of cell suspension and inoculate the side of the mouse, and inoculate 15 mice of each type of cell. Observation and measurement: After cell inoculation, the appearance of tumor and the change of tumor volume were observed every week, and photographed.

Example summary:

(1) This expression vector construction technology has effectively constructed the retroviral vector capable of expressing the hTERT gene:

The pGRN145 plasmid was digested with Hpa I and Not I restriction endonucleases, separated and purified by agar gel electrophoresis to obtain the hTERT cDNA fragment, which was connected with the human retroviral vector pLXSN vector digested with the same restriction endonucleases, and constructed into The expression vector pLXSN-hTERT expresses hTERT efficiently.

(2) Human bone marrow mesenchymal stem cells transduced with pLXSN-hTERT have the advantage of highly expressing hTERT:

The human bone marrow mesenchymal stem cells transduced by this transduction technology have significant ability to express hTERT. The hTERT gene expression in the transduced human bone marrow mesenchymal stem cells was 175±85 ng/10 ⁵ cells/24 hours.

(3) The human bone marrow mesenchymal stem cells transduced by pLXSN-hTERT effectively improved the life cycle of the cells. , cartilage, osteoblast direction differentiation. It has the same differentiation potential as normal human bone marrow mesenchymal stem cells.

Sequences involved in the present invention

<110> Zhejiang University

<120>Method for Transducing Bone Marrow Mesenchymal Stem Cells with Human Telomerase Gene

<160>8

<210>1

<211>25

<212>DNA

<213> Artificial sequence

<220>

<222>(1)...(25)

<223> The upstream primer sequence designed according to the human telomerase gene sequence (GenBank, GI: 109633030)

<400>1

ctctccccct tgaacctaat cgttc

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<222>(1)...(20)

<223> The downstream primer sequence designed according to the human telomerase gene sequence (GenBank, GI: 109633030)

<400>2

aggacacctg gcggaaggag

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<222>(1)...(20)

<223> The upstream primer sequence designed according to the human β-actin gene sequence (GenBank, GI: 5016088)

<400>3

catctcttgc tcgaagtcca

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<220>

<222>(1)...(25)

<223> The downstream primer sequence designed according to the human β-actin gene sequence (GenBank, GI: 5016088)

<400>4

atcatgtttg agaccttcaa ca

Claims (2)

1. A method for human telomerase gene transduction bone marrow mesenchymal stem cells, characterized in that it is realized by the following scheme: (1) Construct the pLXSN-hTERT recombinant expression vector, digest the pGRN145 plasmid with Hpa I and NotI restriction endonucleases, separate and purify the hTERT cDNA fragment by agarose gel electrophoresis, and digest with the same restriction endonucleases The pLXSN vector was connected to construct the pLXSN-hTERT recombinant expression vector; (2) Construct human bone marrow mesenchymal stem cells highly expressing exogenous hTERT gene, according to the instruction manual of LipofectAMINE liposome transduction kit, after preparing the pLXSN-hTERT recombinant expression vector suspension through the amphagocytic packaging cell line PA317, in Human bone marrow mesenchymal stem cells were transduced with pLXSN-hTERT expression vector for 8 hours with the assistance of 1,5-dimethyl-1,5-diazaundecylidene polymethyl bromide to construct high-efficiency expression of exogenous hTERT genetic human bone marrow mesenchymal stem cells; (3) Double selection of human bone marrow mesenchymal stem cells transduced with exogenous hTERT gene was carried out with low concentration of hygromycin, that is, 20 μg/ml hygromycin was used for the first screening of cells for 7 days, and then continued with 50 μg/ml hygromycin. μg/ml hygromycin for a second selection of cells for 7 days. 2. the method for human telomerase gene transduction bone marrow mesenchymal stem cell according to claim 1 is characterized in that: the pLXSN carrier described in step (1) adopts Hpa I and Not I restriction endonuclease enzyme Cut, separated and purified by agarose gel electrophoresis.

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