CN118147232B - Construction method and application of over-expressed TPS1 mesenchymal stem cells - Google Patents
Construction method and application of over-expressed TPS1 mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a construction method and application of an over-expressed TPS1 mesenchymal stem cell. The method comprises the following steps: (1) constructing a recombinant plasmid vector containing TPS1 gene; (2) And transfecting the mesenchymal stem cells by the recombinant plasmid vector to obtain the mesenchymal stem cells over-expressing TPS 1. The invention successfully constructs umbilical cord mesenchymal stem cells (TPS 1-UCMSCs) over-expressing TPS1, solves the problem that exogenous trehalose is difficult to permeate into the mesenchymal stem cells and is expressed continuously, and improves the content of endogenous trehalose expressed by UCMSCs. The TPS1-UCMSCs have higher anti-oxidative stress and anti-apoptosis capacity, higher retention rate on wound surfaces and stronger capacity of treating the wound surfaces.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a mesenchymal stem cell over-expressing TPS1 (trehalose phosphate synthase 1), a construction method and application thereof.
Background
Skin barrier disruption caused by various acute or chronic injury factors such as severe mechanical injury, burns and scalds, vascular dysfunction diseases, tumors and the like is quite common. The existence of the wound surface not only brings the trouble of infection risk and pain, but also can influence the organism function, brings physiological and psychological pains to patients, and increases the economic burden of families and society. Therefore, how to promote rapid healing of wounds remains a significant challenge in the clinic. Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) are a class of adult stem cells with high self-renewal and multipotency, which can be obtained from various tissues such as bone marrow, fat, umbilical cord and amniotic membrane, and are widely used for treating various skin wounds such as full-thickness skin defects, burns and chronic wounds. The therapeutic potential of MSCs is that various cytokines and growth factors secreted by MSCs not only can participate in angiogenesis and cell proliferation, but also can effectively regulate and control local and systemic inflammatory responses, thereby creating a favorable microenvironment for wound healing. But the problems of short survival time, poor cell activity and tolerance and the like of cells in the wound treatment process greatly limit the tissue repair capability of MSCs. Almost all MSCs used are locally disappeared before tissue repair is completed. Therefore, the method for prolonging the survival time of the MSCs and improving the anti-apoptosis capability of the MSCs has important significance for enhancing the tissue repair capability of the MSC.
Disclosure of Invention
The invention aims to provide a construction method and application of an over-expressed TPS1 mesenchymal stem cell so as to enhance the tissue repair capability of the mesenchymal stem cell.
The inventors of the present invention have found in research that although trehalose pretreatment has been reported to protect MSCs from H 2O2 -induced cell viability reduction and apoptosis, and to protect MSCs from uv damage. Mammalian cells lack the mechanism of endogenous synthesis of trehalose and their plasma membranes are impermeable to sugar. The invention uses adenovirus to transfer trehalose phosphate synthase 1 gene (TPS 1 gene) into mesenchymal stem cells for exploring the capacity of trehalose on the inner side of cell membranes to resist apoptosis of MSCs and the action and mechanism of MSCs for treating wound surfaces. Provides thinking for the application of trehalose in improving the survival time and tissue repair capability of MSCs under oxidative stress state, and is favorable for fully exerting the therapeutic value of MSCs in the wound surface with high oxidative stress level.
In order to achieve the above object, a first aspect of the present invention provides a method for constructing mesenchymal stem cells overexpressing TPS1, comprising the steps of:
(1) Constructing a recombinant plasmid vector containing TPS1 genes;
(2) And transfecting the mesenchymal stem cells by the recombinant plasmid vector to obtain the mesenchymal stem cells over-expressing TPS 1.
According to the invention, the sequence of the TPS1 gene is a sequence with the transcript ID of NM134983.3 or a sequence with the transcript ID of NM134983.3, which is obtained by deleting, substituting and adding one or more bases and has the equivalent enzyme activity to TPS1 after expression.
Wherein the TPS1 gene is of Drosophila or Escherichia coli.
The specific source of the mesenchymal stem cells is not particularly limited, and the mesenchymal stem cells may be umbilical cord mesenchymal stem cells (umbilical cords MSCs, UCMSCs), amniotic mesenchymal stem cells, adipose mesenchymal stem cells or bone marrow mesenchymal stem cells.
The present invention may be used to effect transfection of recombinant plasmid vectors using a variety of conventional methods, including but not limited to viral transfection, liposome transfection or electrotransfection.
According to a specific embodiment of the invention, the recombinant plasmid vector is an adenovirus vector, more specifically HBAD-Adeasy-o-TPS1-3 Xflag-EGFP, the transfection is adenovirus transfection, and specific transfection methods can be performed by routine methods in the art.
Further, the detailed steps include:
(1) Cloning the full-length coding sequence of TPS1 gene onto adenovirus vector to obtain adenovirus vector (HBAD-Adeasy-o-TPS 1-3 Xflag-EGFP) with TPS1 over-expression;
(2) Transfecting the adenovirus vector into competent cells, and collecting to obtain TPS1 adenovirus;
(3) Mesenchymal stem cells were inoculated into a cell culture plate and cultured overnight (density of, for example, 3.5X10 4/cm2), the TPS1 adenovirus was transfected into the mesenchymal stem cells (multiplicity of virus infection (MOI), for example, 100), and after 14 hours of transfection, the culture was continued with fresh medium for 34 hours, to obtain mesenchymal stem cells overexpressing TPS 1.
In a second aspect, the present invention provides mesenchymal stem cells obtained by the above-described construction method.
The third aspect of the invention provides the construction method or the application of the mesenchymal stem cells in preparing a reagent for promoting tissue injury repair.
The dosage form of the agent is preferably an external dosage form, such as a paste, a subcutaneous injection, or a spray.
In the reagent, the mesenchymal stem cells can be used alone or can be loaded on a carrier, and the carrier is at least one selected from PBS, hydrogel and graphene.
The methods and mesenchymal stem cells of the invention are particularly useful for promoting repair of acute or chronic wound injury.
The beneficial technical effects of the invention include:
(1) The invention successfully constructs umbilical cord mesenchymal stem cells (TPS 1-UCMSCs) over-expressing TPS1, solves the problem that exogenous trehalose is difficult to permeate into the mesenchymal stem cells and is expressed continuously, and improves the content of endogenous trehalose expressed by UCMSCs.
(2) The TPS1-UCMSCs constructed by the invention have higher anti-oxidative stress and anti-apoptosis capacity.
(3) The TPS1-UCMSCs constructed by the invention have higher retention rate on the wound surface and stronger capability of treating the wound surface.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a diagram showing the construction of an adenovirus vector plasmid for TPS1 prepared in the examples of the present invention.
FIG. 2 shows MOI screening of TPS1 adenovirus transfected UCMSCs prepared in the examples of the present invention; wherein, the A graph is a fluorescence microscope imaging graph of UCMSCs transfected by TPS1 adenovirus, the B graph shows the transfection efficiency of UCMSCs transfected by TPS1 adenovirus, the C graph shows the detection result of trehalose synthase activity of TPS1-UCMSCs, the D graph shows the detection result of trehalose content of intracellular TPS1-UCMSCs, and the E graph shows the detection result of trehalose content of TPS1-UCMSCs in extracellular medium.
FIG. 3 shows the phenotypic change results of TPS1-UCMSCs constructed in the examples of the present invention; wherein, the A graph is a flow cytometry detection graph of NC-UCMSCs and TPS1-UCMSCs, the B graph shows GFP positive rate results of the flow cytometry detection of NC-UCMSCs and TPS1-UCMSCs, and the C graph shows mesenchymal stem cell surface marker results of the flow cytometry detection of NC-UCMSCs and TPS 1-UCMSCs.
FIG. 4 shows the results of anti-apoptotic capacity assays of TPS1-UCMSCs constructed in the examples of the present invention; wherein, the A graph is the detection result of the flow cytometry, and the B graph and the C graph are the corresponding apoptotic cell percentage graph, which respectively correspond to 200 mu M H 2O2 and 0% FBS.
FIG. 5 shows survival time of TPS1-UCMSCs constructed in the examples of the present invention in acute wounds; wherein, the A graph is a mouse living body imaging graph, the B graph is a graph of MSCs retention rate changing along with injection time, and the C graph is a graph of total fluorescence intensity changing along with injection time.
FIG. 6 shows the effect of TPS1-UCMSCs constructed in the examples of the present invention on the wound surface of the full-thickness skin defect of mice; wherein, the A graph is a picture of the wound healing condition of the mice, and the B graph is a time-varying graph of the wound healing rate.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
Example 1:
This example is presented to illustrate the construction of TPS1-UCMSCs
1. Isolation and culture of UCMSCs
The neonatal umbilical cord samples obtained under aseptic conditions were segmented into 2-3 cm pieces, and the arteriovenous and epidermis were removed after 3 washes with sterile PBS. Then, the three-dimensional tissue of 2-3 mm is sheared by sterile ophthalmology and evenly distributed into T75 cell culture flasks. The cells were harvested and passaged at 14 days after 3 days of liquid exchange after standing in serum-free medium dedicated for umbilical mesenchymal stem cells for 7 days. Passage to P3 generation for subsequent experiments.
2. TPS1 adenovirus preparation and MOI screening
Cloning the full-length coding sequence (transcript ID is the sequence shown as NM 134983.3) of TPS1 onto an adenovirus vector to obtain an adenovirus vector HBAD-Adeasy-o-TPS1-3 xflag-EGFP over-expressing TPS1, and collecting TPS1 adenovirus after transfection of competent cells (figure 1). Mesenchymal stem cells were inoculated into a cell culture plate at a density of 3.5X10 4/cm2 for overnight culture, transfected with TPS1 adenovirus having MOI of 10, 30, 100, 300 and 500, and cultured for 14 hours with fresh medium replaced for further 34 hours to obtain mesenchymal stem cells (TPS 1-UCMSCs) overexpressing TPS 1. The infection efficiency was determined by observing green fluorescence expression under a fluorescence microscope. And detecting the trehalose content and the trehalose synthase activity of TPS1-UCMSCs by using a trehalose content detection kit and a trehalose synthase activity detection kit. The results showed that the transfection efficiency of cells was about 80% at moi=100 (fig. 2 a and B, where a is a fluorescence microscopy image of TPS1 adenovirus transfected UCMSCs, and B shows the transfection efficiency of TPS1 adenovirus transfected UCMSCs). Compared to UCMSCs transfected with control empty virus (NC-MSCs), both intracellular and extracellular supernatants had significantly increased trehalose content and intracellular trehalose synthase activity was significantly enhanced (FIGS. 2C and D, where C shows the results of trehalose synthase activity detection of TPS1-UCMSCs and D shows the results of trehalose content detection of TPS 1-UCMSCs). These demonstrate that TPS1 transfection is capable of intracellular synthesis of trehalose synthase (fig. 2D) not only to synthesize trehalose in cells, but also to secrete trehalose synthesized in cells into extracellular medium (fig. 2E). The above results demonstrate that TPS1-UCMSCs capable of expressing and secreting trehalose were successfully constructed.
Example 2:
this example is presented to demonstrate the phenotypic change of TPS1-UCMSCs
1. Morphology of TPS1-UCMSCs
The morphology of cells was observed under an inverted phase contrast microscope after transfection of UCMSCs with TPS1 adenovirus and NC adenovirus with moi=100, and the results showed that the cells were fiber-like, long spindle-shaped, complete in envelope, clear in nucleus, and that the GFP positive rates of NC-UCMSCs and TPS1-UCMSCs were 95.75% and 92.63% respectively by flow cytometry (fig. 3a and B, where a is a flow cytometry detection graph of NC-UCMSCs and TPS1-UCMSCs, and B shows the GFP positive rate results of flow cytometry detection of NC-UCMSCs and TPS 1-UCMSCs.
2. TPS1-UCMSCs phenotype protein
The results of the flow cytometer detecting the mesenchymal stem cell surface markers of NC-UCMSCs and TPS1-UCMSCs show that the positive rates of CD29 and CD105 are more than 99 percent, and the positive rates of CD34 and CD45 are less than 3 percent, which are consistent with the phenotype of the mesenchymal stem cells (the C and C diagrams of FIG. 3 show the results of the flow cytometer detecting the mesenchymal stem cell surface markers of NC-UCMSCs and TPS1-UCMSCs, wherein the abscissa is the channel of the flow cytometer, different fluorescence requires different excitation wavelength and emission wavelength channels, PE, APC and PerCP represent the fluorescent groups on the primary antibody, and the group brackets are the names of the primary antibodies.
Example 3:
This example is used to demonstrate the anti-apoptotic capacity detection of TPS 1-UCMSCs.
Cell transfection was performed in the same manner as above, and after NC-UCMSCs and TPS1-UCMSCs were obtained, 200. Mu.M H 2O2 and 0% FBS were used to interfere with NC-UCMSCs and TPS1-UCMSCs. Cells were then digested with EDTA-free 0.25% trypsin, washed with PBS, centrifuged to harvest the cells and resuspended in flow buffer. Alexa Fluor647/7-AAD apoptosis assay reagent was used followed by Alexa Fluor647 dye followed by 7-AAD dye according to instructions. And finally, detecting apoptosis by using a flow cytometer. The results show that the TPS1-UCMSCs group has lower apoptosis rate under 200. Mu.M of H 2O2 and 0% FBS oxidative stress (FIG. 4, wherein, A is the detection result of a flow cytometer, B and C are corresponding apoptosis cell percentage ratio graphs, which respectively correspond to 200. Mu.M of H 2O2 and 0% FBS).
Example 4:
This example is presented to demonstrate the survival time of TPS1-UCMSCs in acute wounds
6 Male C57B/L6 mice of 6-8 weeks were bred for one week and then modeled as a full-thickness skin defect model. The method comprises the following steps: mice were anesthetized by intraperitoneal injection of 1% pentobarbital at a dose of 40 mg/kg, and then a circular full-thickness skin defect of 1:1 cm diameter was established on the back after depilatory cream removal. NC-UCMSCs and TPS1-UCMSCs were resuspended with 200. Mu.L PBS and injected subcutaneously at 4 points at the wound edge using a 1mL syringe, 50. Mu.L each. Live small animal imaging was performed on days 0,3 and 7. The results show that the cell retention rate of the TPS1-UCMSCs group is higher than that of the NC-UCMSCs group, and the TPS1-UCMSCs group still has higher fluorescence intensity on the 7 th day (FIG. 5, wherein, A is a mouse in vivo imaging graph, B is a graph of MSCs retention rate with injection time, and C is a graph of total fluorescence intensity with injection time).
Example 5:
this example is used to demonstrate the efficacy of TPS1-UCMSCs in treating full-thickness skin defect wounds in mice
Modeling the full-thickness skin defect model, injecting NC-UCMSCs and TPS1-UCMSCs in the same way, photographing and observing on days 0, 3, 5, 7 and 10, and calculating the wound healing rate of the mice. The results show that the TPS1-UCMSCs treatment group has faster wound healing speed (figure 6, wherein, A is a picture of the wound healing condition of mice, and B is a time-dependent picture of the wound healing rate).
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.
Claims (5)
1. Application of mesenchymal stem cells over-expressing TPS1 in preparation of reagent for promoting tissue injury repair, wherein the construction method of the mesenchymal stem cells over-expressing TPS1 comprises the following steps:
(1) Constructing a recombinant plasmid vector containing TPS1 genes;
(2) Transfecting the recombinant plasmid vector into a mesenchymal stem cell to obtain a mesenchymal stem cell over-expressing TPS 1;
the sequence of the TPS1 gene is a sequence shown in NM134983.3 as a transcript ID;
the injury is acute wound injury or chronic wound injury.
2. The use according to claim 1, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells, amniotic mesenchymal stem cells, adipose mesenchymal stem cells or bone marrow mesenchymal stem cells.
3. The use according to claim 1, wherein the method of transfection is selected from viral transfection, liposome transfection or electrotransfection.
4. The use according to claim 1, wherein the construction method comprises the steps of:
(1) Cloning the full-length coding sequence of the TPS1 gene to an adenovirus vector to obtain the adenovirus vector over-expressing TPS 1;
(2) Transfecting the adenovirus vector into competent cells, and collecting to obtain TPS1 adenovirus;
(3) And inoculating the mesenchymal stem cells into a cell culture plate for culturing overnight, and transfecting the mesenchymal stem cells with the TPS1 adenovirus to obtain the mesenchymal stem cells over-expressing TPS 1.
5. The use according to claim 1, wherein the agent is a paste, a subcutaneous injection or a spray;
the reagent further comprises a carrier of mesenchymal stem cells, wherein the carrier is selected from at least one of PBS, hydrogel and graphene.
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