CN105233279A - Glioma holoantigen and preparation method and application thereof - Google Patents
Glioma holoantigen and preparation method and application thereof Download PDFInfo
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- CN105233279A CN105233279A CN201510651466.2A CN201510651466A CN105233279A CN 105233279 A CN105233279 A CN 105233279A CN 201510651466 A CN201510651466 A CN 201510651466A CN 105233279 A CN105233279 A CN 105233279A
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Abstract
The invention relates to a glioma holoantigen and a preparation method and application thereof. The preparation method includes the steps of collecting glioma stem cells and glioma non-stem cells, mixing the cells, and subjecting the mixture of the cells to cell lysis, centrifuging and supernatant removing so as to obtain glioma holoantigen. The glioma holoantigen prepared from the glioma stem cells and glioma non-stem cells carries more complete antigenic information as compared with that of existing glioma holoantigen; therefore, vaccine prepared from the glioma holoantigen can be applied to killing not only the glioma stem cells but also tumor cells of gliomas, and has high tumor killing rate and high immunogenicity.
Description
Technical field
The present invention relates to biological engineering and biomedicine field, particularly full tumor antigen of glioma and preparation method thereof.
Background technology
Glioma derives from neurepithelial tumor, is the modal malignant tumor of intracranial, accounts for the 40-50% of whole intracranial tumor, high with sickness rate, relapse rate is high, fatality rate is high, becomes a difficult problem for oncotherapy.Traditional therapy is mainly performed the operation with chemicotherapy to improve the existence probability of patient.But the glioma being positioned at critical function district is difficult to accomplish full excision.Radiotherapy is almost the conventional therapy of various glioma, but therapeutic evaluation differs, and except medulloblastoma is extremely sensitive to radiotherapy, outside ependymoma medium sensitivity, other types are all insensitive to radiotherapy.Chemotherapeutics is limited to the toxic and side effects of blood brain barrier and medicine, and curative effect is not still affirmed.In recent years, immunization therapy becomes the effective treatment means of cerebral glioma after operation, chemicotherapy.With other mode use in conjunction, specifically have very strong complementary action, the immune system impaired to patient can play unique curative effect of restoration and reconstitution, thus prevents recurrence and the transfer of tumor further.
After carrying out enrichment culture by serum free culture system method to glioma stem cell in prior art, cracking obtains glioma antigen, because this antigen only possesses the antigenic information that glioma stem cells carries, the tumor sample stem cell in glioma and a small amount of tumor cell can only be killed, and owing to there is certain microbial contamination in serum free culture system liquid, and animal sources immunogenicity, Antigen Stability prepared by the glioma stem cell therefore obtained by serum free culture system method is poor, specificity is more weak, therefore the glioma therapeutic vaccine obtained by this antigen has poor stability, specificity is more weak, kill the defects such as the not high and immunogenicity of ratio of outflow is weak.
Summary of the invention
Technical problem to be solved by this invention is, limited for the antigenic information in prior art entrained by glioma antigen, the vaccine obtained by this antigen kills the defects such as ratio of outflow is low, less immunogenic, provides full tumor antigen of glioma that a kind of antigenic information is more complete and preparation method thereof.
The technical solution adopted for the present invention to solve the technical problems is: the preparation method of the full tumor antigen of a kind of glioma, and described preparation method comprises the following steps:
Obtain glioma stem cell and glioma non-stem cell, carry out lysis after both being mixed, namely obtain the full tumor antigen of glioma.
In the preparation method of the full tumor antigen of glioma provided by the invention, the number ratio of glioma stem cell and glioma non-stem cell is 1:2-2:1.
In the preparation method of the full tumor antigen of glioma provided by the invention, the number ratio of glioma stem cell and glioma non-stem cell is 1:1.
In the preparation method of the full tumor antigen of glioma provided by the invention, multigelation cracking process or x-ray bombardment method is adopted to carry out lysis.
In the preparation method of the full tumor antigen of glioma provided by the invention, the condition adopting multigelation cracking process to carry out lysis is: liquid nitrogen or-80 DEG C of frozen and 37 DEG C of water-baths are melted again, multigelation 3 times.
In the preparation method of the full tumor antigen of glioma provided by the invention, before carrying out lysis, also comprise the enrichment culture process adopting serum-free medium to carry out described glioma stem cell and glioma non-stem cell.
In the preparation method of the full tumor antigen of glioma provided by the invention, described serum-free medium is: the culture fluid containing fibroblast growth factor, epithelical cell growth factor and B27.
In the preparation method of the full tumor antigen of glioma provided by the invention, get the glioma stem cell that is in exponential phase and glioma non-stem cell is carrying out lysis.
In the preparation method of the full tumor antigen of glioma provided by the invention, after lysis, also comprise centrifugation step, centrifugal condition is: 1-4 DEG C, the centrifugal 10-20min of 5000rpm.
The present invention also provides a kind of glioma full tumor antigen, obtained by the preparation method of the full tumor antigen of above-mentioned glioma.
The present invention protects the application of the full tumor antigen of above-mentioned glioma in the full tumor antigen vaccine of preparation glioma further.
Implement full tumor antigen of glioma provided by the invention and preparation method thereof, following beneficial effect can be reached: the antigenic information that the more existing glioma antigen of the full tumor antigen of glioma adopting glioma stem cell and glioma non-stem cell to prepare carries is more more complete, therefore the vaccine prepared by the full tumor antigen of glioma provided by the invention is adopted, glioma stem cell can not only be killed, but also glioma tumor cell can be killed, kill ratio of outflow higher, and immunogenicity is stronger.
Detailed description of the invention
The preparation method of the full tumor antigen of a kind of glioma provided by the invention, the method comprises the following steps:
1, acquisition glioma stem cell and glioma non-stem cell carry out enrichment culture respectively;
2, lysis is carried out by after the glioma stem cell after propagation in step 1 and the mixing of glioma non-stem cell;
3, carry out centrifugal to the glioma stem cell after cracking in step 2 and glioma non-stem cell, after abandoning supernatant, namely gather in the crops the full tumor antigen of glioma.
Particularly, in step 1, the obtaining step of glioma stem cell and glioma non-stem cell is:
(11) glioma tumor tissue is obtained;
(12) from the glioma tumor tissue step (11), glioma is obtained unicellular and carry out enrichment culture;
(13) glioma from step (12) after propagation is unicellular isolates glioma stem cell and glioma non-stem cell.
In step (11), get fresh human brain human gliomas, in the present invention, this human gliomas block takes from the fresh specimens of the routine patients with gliomas excision of Huashan Hospital Affiliated To Fudan Univ neurosurgery 11, all makes a definite diagnosis through postoperative pathological.Then, fresh Human Brain Gliomas is used containing 120mmol.L
-1naCI, 5mmol.L
-1kCl, 25mmol.L
-1glucose (glucose) and 20mmol.L
-1blood stains cleaned by the buffer of Pipes (piperazine-Isosorbide-5-Nitrae-two ethyl sulfonic acid), cut into lmm
3the samples of human glioma block of size is for subsequent use.
In step (12), by DMEM culture fluid washing 2-3 time of the glioma tumor tissue of process in step (11), add 0.2% collagenase IV of 5-10 times of tissue volume, 10min is digested in 37 DEG C of incubators, Eddy diffusion cell after the centrifugal 5min of 1000r/min, after removing erythrocyte with erythrocyte cracked liquid, 400 order steel meshes filter, and obtain single cell suspension, single cell suspension is moved into centrifuge tube, after 1000r/min is centrifugal, abandons supernatant.Cell precipitation after centrifugal with serum-free medium Eddy diffusion, preferably, serum-free medium is the DMEM/F12 culture fluid containing 20ng/ μ LbFGF (fibroblast growth factor), 20ng/ μ LEGF (epithelical cell growth factor) and 20%B27; By 3 × 10 after counting
5individual/cm
2density be inoculated in 25cm
2culture bottle, puts 5%CO
2incubator 37 DEG C is hatched.Cover with collecting cell after cell until culture bottle, add 0.2% collagenase IV after centrifugal and digest about 5min, repeatedly blow and beat, supernatant is abandoned after centrifugal, renewed vaccination after suspending, in the ratio Secondary Culture to 120 day of 1:2 or 1:3, can obtain the slender cytosol of the glioma being in exponential phase.
With prior art adopt serum free culture system method cultivate unicellular unlike, in this step, the present invention adopts serum-free medium to carry out enrichment culture to unicellular; Because serum free culture system liquid exists certain microbial contamination, and animal sources immunogenicity and limited source etc., therefore, it is unicellular that the present invention adopts serum-free medium to cultivate, not only can avoid the unfavorable factor that serum free culture system method is brought, provide a more stable condition of culture for unicellular, and unicellular after convenient purification propagation, be convenient to follow-up processed, make the final glioma full tumor antigen stability that obtains and specificity stronger.In addition, it is unicellular in order to obtain abundant glioma for obtaining the unicellular one side of glioma being in exponential phase in this step; On the other hand, the unicellular character of glioma being in exponential phase is more stable, and metabolism is more vigorous, and therefore, the antigenic information obtained entrained by glioma stem cell is more.
In step (13), get the slender cytosol of glioma obtained in step (12) digest through 0.2% collagenase IV, wash, centrifugal after use PBS Eddy diffusion cell, and to adjust cell concentration be l × l0
5individual/ml, CD133/l-PE labelling is carried out by test kit description, the cell going out to be labeled through selected by flow cytometry apoptosis and glioma stem cell, the i.e. glioma non-stem cell be not labeled, collect glioma stem cell respectively and glioma non-stem cell for subsequent use to treat.Glioma non-stem cell in the present invention refers to the glioma tumor cell except glioma stem cell.
These are only a kind of method obtaining glioma stem cell and glioma non-stem cell provided by the invention, be understandable that, the method obtaining glioma stem cell and glioma non-stem cell by other means is all applicable to the present invention.
In step 2, mix than the glioma stem cell and glioma non-stem cell of getting exponential phase in step 1 respectively by the number of 1:2-2:1, rinse with PBS, after centrifugal, abandon supernatant, adopt multigelation cracking process or x-ray bombardment method to carry out cracking to cell.
The condition of multigelation cracking is: liquid nitrogen or-80 DEG C are after frozen one week, melts again in 37 DEG C of water-baths, multigelation 3 times.Cause expansion because the salinity of ice pellets formation and remaining cell liquid in cell increases, make cellularity broken, lose activity.
The condition of x-ray bombardment method is: radiation 2-3 minute under x-ray (6Gray).
After tested, by x-ray bombardment method obtain the full tumor antigen of glioma kill the full tumor antigen of glioma that ratio of outflow will obtain apparently higher than multigelation cracking, therefore, in the present invention, the preferential x-ray bombardment method that adopts carries out cracking to cell.
Be understandable that, the mode of lysis is also not limited thereto, and the method for other lysises is equally applicable to the present invention, as ultrasonication etc.
In step 3, adopt suction pipe repeatedly to blow and beat to the glioma stem cell of cracking and glioma non-stem cell, and 5000rpm low-temperature centrifugation 10-20min obtain mixture at 4 DEG C, i.e. the full tumor antigen of glioma.
The antigenic information of glioma stem cell is not only carried by the full tumor antigen of glioma provided by the invention, and carry the antigenic information of glioma non-stem cell, therefore, relative to glioma antigen of the prior art, antigenic information is more complete, the vaccine prepared by the full tumor antigen of glioma provided by the invention can not only kill glioma stem cell, and a large amount of glioma non-stem cell can be killed, immunogenicity is comparatively strong, and it is higher to kill ratio of outflow.
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
A preparation method for the full tumor antigen of glioma, comprises the following steps:
1, to take the logarithm respectively the glioma stem cell of trophophase and glioma non-stem cell each 5 × 10
7individual;
2, carry out PBS solution cleaning by after the glioma stem cell in step 1 and the mixing of glioma non-stem cell, after centrifugal, remove supernatant;
3, the glioma stem cell in step 2 and glioma non-stem cell are irradiated 2-3 minute to the whole cracking of cell under X-ray (6Gray);
4, after the glioma stem cell after cracking in step 3 and glioma non-stem cell being blown and beaten repeatedly, in-4 DEG C, the mixture that obtains of the centrifugal 15min of 5000rpm is the full tumor antigen of glioma.
The process of the full tumor therapeutic vaccine of glioma prepared by the full tumor antigen of glioma adopting this embodiment 1 to provide is:
1) get 20ml human peripheral, isolate mononuclear cell with Ficon lymphocyte separation medium, press (2.5-5) x10 with after PBS solution washing
7/ 3ml/ hole is inoculated in six orifice plates, and by RPMI-l640 culture medium at 37 DEG C, 5%CO
2incubator in place cultivation 90 minutes, adherent cell collecting, i.e. mononuclear cell.
2) mononuclear cell to be inoculated in the RPMI-l640 culture medium containing 800U/mL grain-macrophage cytokines and 1000U/mL interleukin-4 and at 37 DEG C, CO
2concentration is, after inducing 3-5 days in the incubator of 5%, rock culture bottle, and half amount changes liquid, supplements grain-macrophage cytokines and interleukin-4, maintain grain-macrophage cytokines and interleukin-4 concentration constant, obtain immature DC;
3) after immature DC cultivates 7 days, by PBS solution (PH=7.4) by immature DC Cell sap concentration adjustment to 2 × 10
6individual/ml, gets 10uL immature DC Cell sap and the full tumor antigen Dual culture of 150ug/uL glioma;
4) add 5ng/ml tumor necrosis factor-alpha and 8ng/ml il-1 again and continue to cultivate the ripe and full tumor antigen of load epineural glioma of 18h to DC.
For verifying the remarkable result of the full tumor antigen of glioma provided by the invention further, by detecting the immunogenicity of the full tumor antigen therapeutic vaccine of glioma of preparation in embodiment 1, carry out indirect verification beneficial effect of the present invention.
Experimental subject:
1, experimental group: the glioma therapeutic vaccine of what the embodiment of the present invention 1 provided the carry full tumor antigen of glioma;
2, from the difference of experimental group, matched group: the glioma therapeutic vaccine carrying the glioma antigen obtained by prior art, is only that glioma antigen is different.
The preparation of effector lymphocyte: by 30 6 ~ 8 week age female Babl/c mice be divided into 3 groups at random: experimental group, matched group, often organize 10; Experimental group with concentration for 10
5the vaccine solution of individual/mL is immunogen, matched group equally with concentration for 10
5the vaccine solution of individual/mL is immunogen, performs following operation respectively to two groups of immunogens:
Each group in mice dorsal part root of the tail portion subcutaneous injection immunogen, every only 100 μ L, afterwards at interval of 1 week, in kind booster immunization 1 time, immune 3 times altogether; After final immunization 1 week, disconnected neck puts to death mice, aseptically get spleen, mill with 100 eye mesh screens, collecting cell suspension, be separated splenocyte with Ficoll-Hypaque layering liquid density－gradient centrifuga－tion method, regulate cell density to be 1 × 10 by the RPMI1640 culture medium containing mass percentage concentration being the hyclone of 10%
6individual/mL, as the effector lymphocyte of subsequent experimental.
Test an external tumor of killing to test
Target cell (neuroglial cytoma) is inoculated in (three wells) in 96 orifice plates, cultivates in incubator after 72 hours and use ametycin process.Get the effector lymphocyte after Dual culture by effect target than adding in 96 orifice plates for 5:1,10:1,20:1,40:1,80:l, cultivate after 12 hours with KRP buffer (131.2mmol/LNaCI in incubator, 4.71mmol/LKCI, 2.47mmol/LCaC12, l.24mmol/LMgSO4,2.48mmol/LNa3PO4,10mmol/LHEPES, pH7.4) add lml after process and hatch 12-18 hour containing 0.5 μ C/ml [methyl-3H]-thymidine 37 DEG C, get 3 stopped reactions express developed with the PBS containing 10mmol/L glucose of pre-cooling.The NaOH adding 0.lmmol/L acts on 2 hours, gets cell pyrolysis liquid, and Gamma-5500 liquid flashing counting measures umber of pulse per minute (cpm).
Kill rate is calculated according to formula kill rate (%)=[target matched group cpm mono-(effect target group cpm mono-effect matched group cpm)/target matched group cpm] × 100%.
Result: experimental group has stronger kill capability compared with matched group.When effect target is than 40:1, experimental group vaccine can kill and wound 80.91% scholar 4.79% glioma non-stem cell like cell and 77.32% scholar 4.86% stem cell-like cell, and matched group only can kill and wound 20.3% scholar 6.45% glioma non-stem cell like cell and 40.05% scholar 3.49% stem cell-like cell.
Tumor experiment is killed in experiment disome
Experimental procedure:
Tumor-bearing mice is divided into three groups at random: experimental group, matched group and blank group, blank group root of the tail portion subcutaneous injection normal saline; The existing glioma therapeutic vaccine (10 of matched group root of the tail portion subcutaneous injection
5individual/mL, 0.2mL); Glioma therapeutic vaccine in experimental group root of the tail portion this inventive embodiments 1 of subcutaneous injection (105/mL, 0.2mL) vaccine; Observe the life cycle of each group mice in 60 days.
Result: analyze through life cycle and find: the median survival interval of each experimental group rat is respectively: matched group 21 days, and experimental group is 36 days, blank group 5 days.
Laboratory animal peripheral blood IFN-γ testing result: each group rat peripheral blood IFN-γ concentration is respectively: existing vaccine group 110.29 scholar 7.18pg/ml, experimental group 177.36 scholar 5.95pg/ml, blank group is 77.98 scholar 7.74pg/ml.
Above embodiments of the invention are described; but the present invention is not limited to above-mentioned detailed description of the invention; above-mentioned detailed description of the invention is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protects, also can make a lot of form, these all belong within protection of the present invention.
Claims (10)
1. a preparation method for the full tumor antigen of glioma, is characterized in that: described preparation method comprises the following steps:
Obtain glioma stem cell and glioma non-stem cell, carry out lysis after both being mixed, namely obtain the full tumor antigen of glioma.
2. the preparation method of the full tumor antigen of glioma according to claim 1, is characterized in that, the number ratio of glioma stem cell and glioma non-stem cell is 1:2-2:1.
3. the preparation method of the full tumor antigen of glioma according to claim 2, is characterized in that, the number ratio of glioma stem cell and glioma non-stem cell is 1:1.
4. the preparation method of the full tumor antigen of glioma according to claim 1, is characterized in that, adopts multigelation cracking process or x-ray bombardment method to carry out lysis.
5. the preparation method of the full tumor antigen of glioma according to claim 1, it is characterized in that, before carrying out lysis, also comprise the enrichment culture process adopting serum-free medium to carry out described glioma stem cell and glioma non-stem cell.
6. the preparation method of the full tumor antigen of glioma according to claim 5, is characterized in that, described serum-free medium is: the culture fluid containing fibroblast growth factor, epithelical cell growth factor and B27.
7. the preparation method of the full tumor antigen of glioma according to claim 1, is characterized in that, gets the glioma stem cell that is in exponential phase and glioma non-stem cell is carrying out lysis.
8. the preparation method of the full tumor antigen of glioma according to claim 1, is characterized in that, in described preparation method, after lysis, also comprises centrifugation step, and centrifugal condition is: 1-4 DEG C, the centrifugal 10-20min of 5000rpm.
9. the full tumor antigen of glioma, is obtained by the preparation method of the full tumor antigen of glioma described in any one of claim 1-8.
10. the application of the full tumor antigen of glioma according to claim 9 in the full tumor antigen vaccine of preparation glioma.
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| CN106939298A (en) * | 2017-03-20 | 2017-07-11 | 中国人民解放军第三军医大学 | A kind of full tumour antigen preparation method of glioma and device |
| CN111979197A (en) * | 2019-05-23 | 2020-11-24 | 苏州海苗生物科技有限公司 | Glioma stem cell in-vitro culture method and culture medium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106939298A (en) * | 2017-03-20 | 2017-07-11 | 中国人民解放军第三军医大学 | A kind of full tumour antigen preparation method of glioma and device |
| CN106939298B (en) * | 2017-03-20 | 2019-06-25 | 中国人民解放军第三军医大学 | Method and device for preparing glioma whole tumor antigen |
| CN111979197A (en) * | 2019-05-23 | 2020-11-24 | 苏州海苗生物科技有限公司 | Glioma stem cell in-vitro culture method and culture medium |
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Address after: 518057, room 2, building 10, Shenzhen biological incubation center, No. 302, Nanshan District hi tech, Shenzhen, Guangdong Applicant after: ISTEM REGENERATIVE MEDICINE SCI-TECH CO., LTD. Address before: 518057, Guangdong Province, Nanshan District high tech Zone, South Ring Road, No. 29, No. 02 building, international students, building No. 15 Applicant before: ISTEM REGENERATIVE MEDICINE SCI-TECH CO., LTD. |
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