Method for preparing mesenchymal stem cells expressing insulin in response to glucose changes

Abstract

The present application relates to the technical field of diabetes treatment, and discloses a preparation method of mesenchymal stem cells expressing insulin in response to glucose change, which comprises the following steps: firstly, cloning and screening a promoter capable of regulating gene expression in response to glucose change; then, constructing a lentivirus vector of insulin fusion gene expressing in response to glucose change, replacing the original promoter on the lentivirus vector with the screened promoter to drive the expression of the downstream insulin fusion gene; next, transfecting 293T cells with the constructed lentivirus vector and a packaging vector to complete lentivirus packaging, collecting cell culture solution containing lentivirus, culturing mesenchymal stem cells with the cell culture solution, allowing the lentivirus to infect the mesenchymal stem cells, and screening and diluting the screened single clone cells by using puromycin, so as to finally obtain mesenchymal stem cells expressing insulin in response to glucose change. The prepared mesenchymal stem cells can simulate the expression of insulin in response to blood glucose change in human and animal bodies, and provide a new means for the treatment of diabetes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of diabetes treatment, and in particular to a mesenchymal stem cell preparation method for expressing insulin in response to glucose changes. BACKGROUND

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar, which can cause a variety of serious complications in the late stage, and brings a huge burden to the health and medical resources of patients. At present, the treatment of type I diabetes and advanced type II diabetes mainly depends on insulin injection, but it is difficult to prevent complications. With the development of stem cell biology and cell therapy methods, the use of embryonic stem cells or induced pluripotent stem cells to differentiate into pancreatic islet cells to treat diabetes has the problems of tumorigenicity, immune rejection, low differentiation efficiency, high cost, etc.; while using allogeneic or autologous adult mesenchymal stem cells to treat diabetes can avoid tumorigenicity and immune rejection, but still faces many difficulties, such as poor effect of direct injection of mesenchymal stem cells, low efficiency of mesenchymal stem cells to differentiate into pancreatic islet cells and complicated and expensive technology, and forced expression of insulin to treat diabetes also has the difficulties of unstable insulin expression or gene silencing.

[0003] Patent No. ZL202110434985.9 discloses a method for stably expressing insulin that can be secreted in mesenchymal stem cells. This method uses fusion protein expression technology and 2A self-cleavage technology to establish a method for stably and secretively expressing insulin in mesenchymal stem cells, solving the problem of the inability of mesenchymal stem cells to stably express and secrete insulin. However, it cannot express and secrete insulin in response to changes in glucose concentration, and continuous high secretion may lead to hypoglycemic coma and other shortcomings.

[0004] Therefore, the present application provides a mesenchymal stem cell preparation method for expressing insulin in response to glucose changes to solve the above technical problems. SUMMARY

[0005] The present application provides a mesenchymal stem cell preparation method for expressing insulin in response to glucose changes, aiming to solve the problems of the inability of existing mesenchymal stem cells to express and secrete insulin in response to changes in glucose concentration, low efficiency of mesenchymal stem cells to differentiate into pancreatic islet cells, and easy gene silencing by directly expressing exogenous insulin genes, and provides new drugs and new methods for mesenchymal stem cell treatment of diabetes (including type I and type II advanced diabetes).

[0006] First of all, it should be pointed out that the abbreviations and key terms involved in the present application are defined as follows:

[0007] PKLR: Pyruvate kinase L / R (pyruvate kinase L / R);

[0008] FASN / FAS: Fatty acid synthase

[0009] ACACA: Acetyl-CoA carboxylase alpha

[0010] MLXIPL: MLX interacting protein like

[0011] hInsulin: human Insulin

[0012] mInsulin: mouse Insulin

[0013] mCherry: red fluorescent protein

[0014] pLV: lentiviral plasmid

[0015] The application provides a mesenchymal stem cell preparation method for expressing insulin in response to glucose change.

[0016] (1) The method for driving the expression of human and mouse insulin genes by using a promoter for regulating gene expression in response to glucose concentration change, including a fatty acid synthase promoter (FASN), an acetyl-CoA carboxylase enzyme promoter (ACACA), and a carbohydrate response element binding protein promoter (MLXIPL or CHREBP);

[0017] (2) The method for driving the expression of an mCHerry-2A-Insulin insulin fusion gene by using a promoter for regulating gene expression in response to glucose concentration change; the promoters include a fatty acid synthase promoter (FASN), an acetyl-CoA carboxylase enzyme promoter (ACACA), and a carbohydrate response element binding protein promoter (MLXIPL or CHREBP);

[0018] (3) The preparation process of mesenchymal stem cells for expressing and secreting insulin in response to glucose concentration change, including transfection, virus packaging, transduction, cell screening, and a method for detecting insulin expression and secretion.

[0019] The application is realized through the following scheme.

[0020] The mesenchymal stem cell preparation method for expressing insulin in response to glucose change provided by the application comprises the following steps.

[0021] S1. Cloning and screening of promoters that can regulate gene expression in response to glucose changes:

[0022] S1.1. Determination of promoters that regulate gene expression in response to glucose concentration: By consulting literature, it was found that the expression of genes such as pyruvate kinase L / R (PKLR), fatty acid synthase (FASN or FAS), acetyl-CoA carboxylase alpha (ACACA), and carbohydrate response element binding protein (CHREBP, also known as MLX interacting protein like (MLXIPL)) are affected by glucose concentration or carbohydrate metabolism; subsequently, the promoter sequences of these genes were downloaded from the eukaryotic promoter database.

[0023] S1.2. Primer design and PCR amplification of promoters: Design and synthesize primers for polymerase chain reaction (PCR) amplification of the promoters of the above four genes with the help of a biological company; at the same time, HEK293T cells were cultured and used for extraction of genomic DNA;

[0024] The PCR reaction system includes: 50 ng of genomic DNA, 1.0 μL of PCR amplification forward and reverse primers (25 μM) each, 25 μL of Phanata Max PCR 2 reaction mixture (Novozyme), and water to make up to 50 μL;

[0025] The PCR reaction conditions are: denaturation at 95℃ for 5 min; then denaturation at 95℃ for 1 min, annealing at 55℃ for 20 sec, extension at 72℃ for 1 min, for a total of 35 cycles; finally, extension at 72℃ for 10 min;

[0026] Among them, the PCR products were identified by agarose gel electrophoresis.

[0027] S1.3. Cloning of promoter-driven reporter genes: PKLR, FASN, ACACA, and MLXIPL promoters were loaded into the reporter gene expression vector pGL3-Basic to drive the expression of downstream luciferase genes; gel electrophoresis showed that these promoters were loaded into the reporter gene vector.

[0028] S1.4, screening the promoter responding to glucose regulation reporter gene expression: first, determine the minimum glucose concentration of 0.3 mg / mL to maintain the normal growth of HeLa cells, which is sufficient to maintain the normal growth of HeLa cells;

[0029] Then, the reporter gene expression vector driven by the above-mentioned promoter is transfected into HeLa cells cultured in different sugar concentrations, and at the same time, the promoter (Adenovirus major late promoter: AdML) which does not respond to glucose concentration regulation gene expression is used as a negative control, and the cells are collected after 48 hours, the cell lysate is centrifuged, the supernatant is reserved and used for luciferase activity detection;

[0030] Subsequently, statistical analysis is completed on the test data; the results show that the FASN, ACACA and MLXIPL promoters increase the expression of luciferase gene with the increase of glucose concentration, while the PKLR promoter cannot respond to the change of glucose to regulate the expression of luciferase.

[0031] S2, preparation of bone marrow mesenchymal stem cells secreting mouse and human insulin in response to glucose change:

[0032] S2.1, construction of lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose change: using recombinant ligation method, FASN, ACACA and MLXIPL promoters are respectively replaced with EF1α promoter in pLV-EF1α-mCherry lentiviral vector to drive the expression of downstream mCHerry-2A-hInsulin-His fusion gene, and PCR detection results show that the above-mentioned promoters have replaced the EF1α promoter;

[0033] At the same time, mouse pancreatic tissue and PCR amplified mouse insulin gene are used to replace hInsulin in the above-mentioned mCherry-2A-hInsulin-His to form mCherry-2A-mInsulin-His fusion gene and express it driven by the promoter responding to glucose change; PCR detection results show that mInsulin has replaced hInsulin.

[0034] S2.2, transfection, screening and preparation of bone marrow mesenchymal stem cells secreting mouse insulin in response to glucose change:

[0035] (a) the lentivirus packaging is completed by transfecting the lentivirus vector constructed in the above-mentioned step S2.1 to express the mouse insulin fusion gene driven by the glucose change into 293T cells with packaging vectors pH1 and pH2, and the cell culture liquid containing lentivirus is collected after 72 hours, filtered through a 0.45 μm filter and reserved;

[0036] (b) Culturing mouse bone marrow mesenchymal stem cells in 12-well plates, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with the culture solution containing the lentivirus so as to infect the bone marrow mesenchymal stem cells with the lentivirus;

[0037] (c) After 48 h of infection, screening with puromycin for 48 h, and then diluting a small amount of cells in a 96-well plate to screen for monoclonal cells; determining the expression levels of insulin driven by different promoters by immunoblotting; the results show that the FASN and MLXIPL promoters can drive the expression of mouse insulin (mInsulin) significantly stronger than the ACACA promoter;

[0038] (d) Culturing the insulin-expressing mesenchymal stem cells in culture solutions containing different concentrations of glucose, and after 48 h, collecting the cells and detecting the expression of insulin in the cells under different glucose concentrations by immunoblotting analysis; the results show that the expression of mouse insulin driven by the FASN and MLXIPL promoters increases with the increase of the glucose concentration, while the expression of insulin driven by the ACACA promoter is low and is not significantly affected by the glucose concentration. By analyzing the insulin secretion in the culture solution, it is found that insulin is detected in the culture solution, indicating that the expressed insulin can be successfully secreted outside the cells.

[0039] S2.3, Transfection, Screening and Preparation of Bone Marrow Mesenchymal Stem Cells Secreting Human Insulin:

[0040] (a) Transfecting the 293T cells with the above-constructed lentivirus vector and packaging vectors pH1 and pH2 for the expression of human insulin fusion gene driven by the glucose change-responsive promoter to complete lentivirus packaging, and after 72 h, collecting the cell culture solution containing the lentivirus, filtering it through a 0.45 μm filter and storing it for later use;

[0041] (b) Culturing human bone marrow mesenchymal stem cells in 12-well plates, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with the culture solution containing the lentivirus so as to infect the bone marrow mesenchymal stem cells with the lentivirus;

[0042] (c) After 48 h of infection, screening with puromycin for 72 h, and then diluting a small amount of cells in a 96-well plate to screen for monoclonal cells. Determining the expression levels of insulin driven by different promoters by immunoblotting; the results show that the FASN and MLXIPL promoters can drive the expression of human insulin (hInsulin) significantly stronger than the ACACA promoter, and the latter drives the expression of insulin at a very low level;

[0043] (d) the high insulin expression mesenchymal stem cells are cultured in culture solution containing different glucose concentrations, after 48 hours, the cells are collected, and the expression of insulin in the cells under different glucose concentrations is detected by immunoblotting analysis; the results show that the expression of human insulin driven by FASN and MLXIPL promoters increases with the increase of glucose concentration. By analyzing the insulin secretion in the culture solution, the results show that insulin is detected in the culture solution, indicating that the expressed insulin can be successfully secreted into the extracellular.

[0044] Compared with the related art, the mesenchymal stem cells expressing insulin in response to glucose change provided by the application have the following beneficial effects:

[0045] The mesenchymal stem cells expressing insulin prepared by the application can overcome many problems existing in the current mesenchymal stem cell treatment. First, the problem that mesenchymal stem cells cannot stably express and secrete insulin is solved; second, compared with the existing mesenchymal stem cells that can secrete insulin but cannot secrete in response to glucose change, the mesenchymal stem cells prepared by the application can simulate the expression of insulin in response to blood glucose change in human and animal bodies, avoiding the dangerous situation of hypoglycemic coma caused by continuous high secretion.

[0046] The application has clinical transformation potential, and once successfully applied in the clinic, it can treat type I diabetes and late stage type II diabetes patients, not only bringing economic benefits to the country and region, but also reducing the national medical burden, and bringing new hope to diabetes patients. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is an agarose gel electrophoresis result graph of the promoter PCR product in the application;

[0048] Figure 2 It is an enzyme digestion identification result graph of the promoter cloned in the reporter gene expression vector in the application;

[0049] Figure 3 It is a result graph of the influence of glucose concentration on the expression of the reporter gene driven by the promoter in the application, wherein a, b, c and d respectively represent the influence of different glucose concentrations on the expression activity of luciferase driven by PKLR, FASN, ACACA and MLXIPL promoters, and ADML is an adenovirus promoter which does not contain a carbohydrate response element;

[0050] Figure 4 It is an electrophoresis result graph of the promoter cloned in the pLV-mCHerry-2A-hInsulin-His lentiviral expression vector for detecting the expression of the gene in response to glucose concentration regulation in the application;

[0051] Figure 5Figure 1 is an electrophoretogram of PCR detection of cloned mouse insulin genes (mInsulin, mINS) driven by different promoters in the present application;

[0052] Figure 6 Figure 2 is an immunoblotting result diagram of the influence of different glucose concentrations on the expression of mouse insulin genes driven by different promoters in mouse bone marrow mesenchymal stem cells, (a) immunoblotting detection of the expression of mouse insulin genes driven by different promoters in mouse bone marrow mesenchymal stem cells; (b-d) immunoblotting detection of the expression of mouse insulin genes driven by FASN, ACACA and MLXIPL promoters in mouse bone marrow mesenchymal stem cells under different glucose concentrations; (e) immunoblotting analysis of insulin secretion in cell culture medium.

[0053] Figure 7 Figure 3 is an immunoblotting result diagram of the influence of different glucose concentrations on the expression of human insulin genes driven by different promoters in human bone marrow mesenchymal stem cells, (a) immunoblotting detection of the expression of human insulin genes driven by different promoters in mouse bone marrow mesenchymal stem cells; (b and c) immunoblotting detection of the expression of mouse insulin genes driven by FASN and MLXIPL promoters in mouse bone marrow mesenchymal stem cells under different glucose concentrations; (d) immunoblotting analysis of human insulin secretion in cell culture medium. DETAILED DESCRIPTION

[0054] The present application will be further described below in conjunction with the drawings and embodiments.

[0055] The present application provides a preparation method of mesenchymal stem cells expressing insulin in response to glucose changes, comprising the following steps:

[0056] S1, cloning and screening promoters that can regulate gene expression in response to glucose changes;

[0057] S1.1, determination of glucose concentration-regulated gene expression promoter: by consulting literature, it is found that the expression of pyruvate kinase (Pyruvate kinase L / R, PKLR), fatty acid synthase (Fatty acid synthase, FASN or FAS), acetyl-CoA carboxylase alpha (Acetyl-CoA carboxylase alpha, ACACA) and carbohydrate response element binding protein (Carbohydrate response element binding protein, CHREBP, also known as MLX interacting protein like (MLXIPL)) and other genes are affected by glucose concentration or carbohydrate metabolism; then the promoter sequences of these genes are downloaded from the eukaryotic promoter database.

[0058] S1.2, primer design and PCR amplification of promoter: design and synthesize primers for polymerase chain reaction (PCR) amplification of the promoters of the above four genes with the help of biological companies; at the same time, HEK293T cells are cultured and used for extraction of genomic DNA;

[0059] The PCR reaction system comprises: 50 ng of genomic DNA, 1.0 μL of PCR amplification forward and reverse primers (25 μM) respectively, 25 μL of Phanata Max PCR 2 reaction mixture (Novozyme), and water to make up to 50 μL;

[0060] The PCR reaction conditions are: denaturation at 95℃ for 5 min; then denaturation at 95℃ for 1 min, annealing at 55℃ for 20 sec, extension at 72℃ for 1 min, a total of 35 cycles; finally, extension at 72℃ for 10 min;

[0061] The PCR product is identified by agarose gel electrophoresis (as shown in Figure 1 ).

[0062] S1.3, promoter-driven reporter gene cloning: the PKLR, FASN, ACACA and MLXIPL promoters are loaded into the reporter gene expression vector pGL3-Basic to drive the expression of the downstream luciferase gene; gel electrophoresis shows that these promoters are loaded into the reporter gene vector (as shown in Figure 2 ).

[0063] S1.4, screening of glucose-regulated reporter gene expression promoter: first, determine the minimum glucose concentration of 0.3 mg / mL for maintaining normal growth of HeLa cells, which is sufficient to maintain normal growth of HeLa cells;

[0064] Then, the reporter gene expression vectors driven by the above promoters were transfected into HeLa cells cultured in different sugar concentrations, and the promoter (Adenovirus major late promoter: AdML) which does not respond to the glucose concentration to regulate gene expression was used as a negative control. After 48 hours, the cells were collected, the cell lysate was centrifuged, the supernatant was reserved and used for luciferase activity detection;

[0065] Subsequently, statistical analysis was performed on the test data; the results showed that the FASN, ACACA and MLXIPL promoters increased the expression of luciferase gene with the increase of glucose concentration (see attached Figure 3 Fig. 2), while the PKLR promoter could not respond to the change of glucose to regulate the expression of luciferase.

[0066] S2, preparation of bone marrow mesenchymal stem cells secreting mouse and human insulin in response to glucose changes:

[0067] S2.1, construction of lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose changes: using recombinant ligation method, FASN, ACACA and MLXIPL promoters were replaced with EF1α promoter in pLV-EF1α-mCherry lentiviral vector to drive the expression of downstream mCHerry-2A-hInsulin-His fusion gene. PCR detection results showed that the above promoters had replaced the EF1α promoter (as shown in attached Figure 4 Fig. 3);

[0068] At the same time, mouse pancreatic tissue and PCR amplified mouse insulin gene were used to replace hInsulin in the above mCherry-2A-hInsulin-His to form mCherry-2A-mInsulin-His fusion gene and express under the driving of glucose-responsive promoter; PCR detection results showed that mInsulin had replaced hInsulin (as shown in attached Figure 5 Fig. 4).

[0069] S2.2, transfection, screening and preparation of bone marrow mesenchymal stem cells secreting mouse insulin in response to glucose changes:

[0070] (a) The lentivirus packaging was completed by transfecting the lentivirus vector constructed in the above step S2.1 to express mouse insulin fusion gene in response to glucose changes and packaging vectors pH1 and pH2 into 293T cells. After 72 hours, the cell culture solution containing lentivirus was collected, filtered through a 0.45 μm filter and reserved for use;

[0071] (b) Culturing mouse bone marrow mesenchymal stem cells in 12-well plates, when the cells grow to 60% confluence, using the culture solution containing lentivirus to culture mesenchymal stem cells so that the lentivirus infects the bone marrow mesenchymal stem cells;

[0072] (c) After 48h of infection, using puromycin to screen for 48h, then, diluting a small amount of cells in a 96-well plate to screen for monoclonal cells; using immunoblotting to determine the levels of insulin expressed by different promoters; the results show that the FASN and MLXIPL promoters can drive the expression of mouse insulin (mInsulin) significantly stronger than the ACACA promoter (as shown in FIG. 2a); Figure 6

[0073] (d) Culturing the insulin-expressing mesenchymal stem cells in culture solution containing different concentrations of glucose, after 48h, collecting the cells, and using immunoblotting to analyze the expression of insulin in the cells under different glucose concentrations; the results show that the expression of mouse insulin driven by the FASN and MLXIPL promoters increases with the increase of glucose concentration, while the expression of insulin driven by the ACACA promoter is low and is not significantly affected by the glucose concentration (as shown in FIG. 2b-d). Figure 6 Figure 6 (e) By analyzing the insulin secretion in the culture solution, the results show that insulin is detected in the culture solution (as shown in FIG. 2e), indicating that the expressed insulin can be successfully secreted into the extracellular space.

[0074] S2.3, Transfection, screening, and preparation of bone marrow mesenchymal stem cells secreting human insulin:

[0075] (a) Transfecting the above-mentioned lentivirus vector constructed in step S2.1 to respond to the change of glucose to drive the expression of human insulin fusion gene and the packaging vectors pH1 and pH2 into 293T cells to complete lentivirus packaging, collecting the cell culture solution containing lentivirus after 72h, filtering it through a 0.45 μm filter, and reserving it for use;

[0076] (b) Culturing human bone marrow mesenchymal stem cells in 12-well plates, when the cells grow to 60% confluence, using the culture solution containing lentivirus to culture mesenchymal stem cells so that the lentivirus infects the bone marrow mesenchymal stem cells;

[0077] (c) After 48h of infection, using puromycin to screen for 72h, then, diluting a small amount of cells in a 96-well plate to screen for monoclonal cells. Immunoblotting is used to determine the levels of insulin expressed by different promoters; the results show that the FASN and MLXIPL promoters can drive the expression of human insulin (hInsulin) significantly stronger than the ACACA promoter, and the latter drives the expression of insulin at a very low level (as shown in FIG. 3a); Figure 7

[0078] ​​​(d) The high insulin expressing mesenchymal stem cells were cultured in medium with different glucose concentrations, and after 48 hours, the cells were collected and the expression of insulin in the cells was detected by immunoblotting analysis. The results showed that the expression of human insulin driven by FASN and MLXIPL promoters increased with the increase of glucose concentration (as shown in Figs. 7b and 7c). By analyzing the insulin secretion in the medium, the results showed that insulin was detected in the medium (as shown in Fig. 7d), indicating that the expressed insulin could be successfully secreted into the extracellular. Figure 7 b and 7c). By analyzing the insulin secretion in the medium, the results showed that insulin was detected in the medium (as shown in Fig. 7d), indicating that the expressed insulin could be successfully secreted into the extracellular. Figure 7 d).

[0079] It should be noted that mesenchymal stem cells can have different names according to their organ or tissue sources, such as bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental mesenchymal stem cells, adipose tissue mesenchymal stem cells, and muscle mesenchymal stem cells, but all have the characteristics of mesenchymal stem cells and can differentiate into osteoblasts, chondroblasts, adipocytes, etc. Although the present application uses human and mouse bone marrow mesenchymal stem cells, other mesenchymal stem cells and the present application technology can also have transformation potential. Therefore, the use of mesenchymal stem cells from other tissues and the present application technology to prepare insulin expressing and secreting mesenchymal stem cells is also within the scope of the present application.

[0080] The above description is only an embodiment of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, as described in the specification and drawings of the present application, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing mesenchymal stem cells expressing insulin in response to glucose changes, characterized by, The method comprises the following steps: S1, cloning and screening promoters that can regulate gene expression in response to glucose changes; S1.1, determination of promoters that regulate gene expression in response to glucose concentration; S1.2, primer design and PCR amplification of promoters; S1.3, cloning of promoter-driven reporter genes; S1.4, screening of promoters that regulate reporter gene expression in response to glucose; S2, preparation of bone marrow mesenchymal stem cells that secrete mouse and human insulin in response to glucose changes: S2.1, construction of a lentiviral vector expressing a human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose changes; S2.2, transfection, screening and preparation of bone marrow mesenchymal stem cells that secrete mouse insulin in response to glucose changes; S2.3, transfection, screening and preparation of bone marrow mesenchymal stem cells that secrete human insulin; The determination of promoters that regulate gene expression in response to glucose concentration in step S1.1 is as follows: by consulting literature, it is found that the expression of pyruvate kinase PKLR, fatty acid synthase FASN, acetyl-CoA carboxylase alpha ACACA and carbohydrate response element binding protein MLXIPL genes is affected by glucose concentration or carbohydrate metabolism; then the promoter sequences of these genes are downloaded from the eukaryotic promoter database; The specific steps of primer design and PCR amplification of promoters in step S1.2 are as follows: design and synthesize primers for polymerase chain reaction (PCR) amplification of the promoters of the four genes by a biological company; at the same time, HEK293T cells are cultured and used for genomic DNA extraction; Wherein The PCR reaction system comprises: 50 ng of genomic DNA, 25 μM of PCR amplification forward and reverse primers each 1.0 μL, Phanata Max PCR 2 reaction mixture 25 μL, and water to make up to 50 μL; the PCR reaction conditions are: denaturation at 95℃ for 5 min; then denaturation at 95℃ for 1 min, annealing at 55℃ for 20 sec, extension at 72℃ for 1 min, a total of 35 cycles; finally, extension at 72℃ for 10 min; wherein the PCR products are identified by agarose gel electrophoresis; The specific steps of cloning of promoter-driven reporter genes in step S1.3 are as follows: the PKLR, FASN, ACACA and MLXIPL promoters are loaded into the reporter gene expression vector pGL3-Basic to drive the expression of the downstream luciferase gene; The specific steps of screening the promoter responding to the expression of the glucose-regulated reporter gene in step S1.4 are as follows: first, the minimum glucose concentration of 0.3 mg / mL is determined to be the minimum glucose concentration for maintaining normal growth of HeLa cells, and the glucose concentration is sufficient to maintain normal growth of HeLa cells; then, the reporter gene expression vectors driven by the above-mentioned promoters are transfected into HeLa cells cultured in different glucose concentrations, and at the same time, the promoter AdML not responding to the glucose concentration-regulated gene expression is used as a negative control, and after 48 hours, the cells are collected, the cell lysate is centrifuged, the supernatant is reserved and used for luciferase activity detection; then, statistical analysis is performed on the detection data; In step S2.3, the FASN and MLXIPL promoters are screened, which can drive mouse or human insulin expression.

2. The method for preparing mesenchymal stem cells that express insulin in response to glucose changes as described in claim 1, characterized in that, The specific steps of constructing the lentiviral vector for expressing the human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene responding to glucose change in step S2.1 are as follows: The FASN, ACACA and MLXIPL promoters are respectively replaced with the EF1α promoter in the pLV-EF1α-mCherry lentiviral vector to drive the expression of the downstream mCHerry-2A-hInsulin-His fusion gene; At the same time, the mouse pancreas tissue is used to amplify the mouse insulin gene by PCR, and the obtained gene is used to replace the hInsulin in the above-mentioned mCherry-2A-hInsulin-His to form the mCherry-2A-mInsulin-His fusion gene and drive the expression of the fusion gene responding to the glucose change.

3. The method for preparing mesenchymal stem cells that express insulin in response to glucose changes as described in claim 2, characterized in that, The specific steps of transfecting, screening and preparing the bone marrow mesenchymal stem cells secreting mouse insulin responding to glucose change in step S2.2 are as follows: (a) The lentivirus packaging is completed by transfecting the lentiviral vector for expressing the mouse insulin fusion gene responding to glucose change constructed in step S2.1 and the packaging vectors pH1 and pH2 into 293T cells, and after 72 hours, the cell culture solution containing the lentivirus is collected, filtered through a 0.45 μm filter and reserved; (b) The mouse bone marrow mesenchymal stem cells are cultured in a 12-well plate, and when the cells grow to 60% confluence, the mesenchymal stem cells are cultured with the culture solution containing the lentivirus so as to infect the bone marrow mesenchymal stem cells with the lentivirus; (c) After 48 hours of infection, the puromycin is used for screening for 48 hours, and then a small amount of cells is diluted in a 96-well plate to screen the single clone cells; the immunoblotting method is used to determine the expression level of insulin under different promoters; (d) The mesenchymal stem cells expressing insulin are cultured in a culture solution containing different concentrations of glucose, and after 48 hours, the cells are collected and subjected to immunoblotting analysis to detect the expression of insulin under different glucose concentrations.

4. The method for preparing mesenchymal stem cells that express insulin in response to glucose changes as described in claim 2, characterized in that, The specific steps of transfecting, screening and preparing the bone marrow mesenchymal stem cells secreting human insulin in step S2.3 are as follows: (a) Transfect the 293T cells with the constructed lentivirus vector and packaging vector pH1 and pH2 in step S2.1 above to complete the lentivirus packaging, collect the cell culture solution containing the lentivirus after 72 hours, filter through a 0.45 μm filter and reserve; (b) Culture the human bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culture the mesenchymal stem cells with the culture solution containing the lentivirus to infect the mesenchymal stem cells with the lentivirus; (c) After 48 hours of infection, screen with puromycin for 72 hours, then dilute a small amount of cells in a 96-well plate to screen the monoclonal cells, and determine the expression level of insulin expressed by different promoters by immunoblotting; (d) Culture the mesenchymal stem cells with high expression of insulin in culture solution containing different concentrations of glucose, collect the cells after 48 hours, and detect the expression of insulin in the cells under different glucose concentrations by immunoblotting analysis.

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