Preparation method of mesenchymal stem cells for expressing insulin in response to glucose change

Abstract

The invention relates to the technical field of diabetes treatment, and discloses a preparation method of mesenchymal stem cells for expressing insulin in response to glucose change, and the method comprises the following steps: cloning and screening out a promoter capable of regulating gene expression in response to glucose change; then constructing a lentiviral vector for expressing the insulin fusion gene in response to glucose change, and replacing the original promoter on the lentiviral vector with the screened promoter to drive the expression of the downstream insulin fusion gene; transfecting 293T cells with the constructed lentiviral vector and packaging vector to complete lentivirus packaging, collecting a lentivirus-containing cell culture solution, culturing the mesenchymal stem cells by using the lentivirus-containing cell culture solution to enable the lentivirus to infect the mesenchymal stem cells, and screening and diluting and screening monoclonal cells by using puromycin to obtain the lentivirus-containing mesenchymal stem cells. And finally obtaining the mesenchymal stem cells for expressing insulin in response to glucose change. The mesenchymal stem cells prepared by the invention can simulate the change of blood glucose in human and animal bodies to express insulin, and a new means is provided for the treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of diabetes treatment, and in particular to a method for preparing mesenchymal stem cells that express insulin in response to changes in glucose. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia. In the late stage, it can cause a variety of serious complications, which imposes a huge burden on patients' health and medical resources. At present, the treatment of type 1 diabetes and late type 2 diabetes mainly relies on insulin injection, but it is difficult to prevent the occurrence of complications. With the development of stem cell biology and cell therapy, the use of embryonic stem cells or induced pluripotent stem cells to differentiate into pancreatic islet cells to treat diabetes has problems such as tumorigenicity, immune rejection, low differentiation efficiency, and high cost; and the use of allogeneic or autologous adult mesenchymal stem cells to treat diabetes can avoid tumorigenicity and immune rejection, but still faces many difficulties, such as the poor effect of direct injection of mesenchymal stem cells, the low efficiency of mesenchymal stem cell differentiation into pancreatic islet cells, the cumbersome technology and high price, and the difficulties of forced expression of insulin to treat diabetes, such as unstable insulin expression or gene silencing.

[0003] The invention patent with the patent number ZL202110434985.9 discloses a method for stably expressing secretable insulin in mesenchymal stem cells. This method uses fusion protein expression technology and 2A self-cleavage technology to establish a method for stably expressing and secreting insulin in mesenchymal stem cells, solving the problem that mesenchymal stem cells cannot stably express and secrete insulin. However, it has the disadvantages of not being able to express and secrete insulin in response to changes in glucose concentration, and if the secretion is high, it may cause hypoglycemia coma.

[0004] Therefore, the present invention provides a method for preparing mesenchymal stem cells that express insulin in response to changes in glucose, in order to solve the above technical problems. Summary of the invention

[0005] The present invention provides a method for preparing mesenchymal stem cells that express insulin in response to changes in glucose, aiming to solve the problems of existing mesenchymal stem cells in the treatment of diabetes, namely, the inability to express and secrete insulin in response to changes in glucose concentration, the low efficiency of mesenchymal stem cells in differentiating into pancreatic islet cells, and the easy occurrence of gene silencing by directly expressing exogenous insulin genes, thereby providing new drugs and methods for the treatment of diabetes (including late-stage type I and II diabetes) with mesenchymal stem cells.

[0006] It should be noted first that the definitions of abbreviations and key terms involved in the present invention are:

[0007] PKLR: Pyruvate kinase L / R;

[0008] FASN / FAS: fatty acid synthase;

[0009] ACACA: Acetyl-CoAcarboxylase 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 method for preparing mesenchymal stem cells that express insulin in response to changes in glucose provided by the present invention has the following key technical points:

[0016] (1) A method for driving the expression of human and mouse insulin genes using promoters that regulate gene expression in response to changes in glucose concentration, including the lipase synthase promoter (FASN), the acetyl-CoA carboxylase promoter (ACACA), and the carbohydrate response element binding protein promoter (MLXIPL or CHREBP);

[0017] (2) A method for driving the expression of the mCHerry-2A-Insulin insulin fusion gene using promoters that regulate gene expression in response to changes in glucose concentration; these promoters include the lipase promoter (FASN), the acetyl-CoA carboxylase promoter (ACACA), and the carbohydrate response element binding protein promoter (MLXIPL or CHREBP);

[0018] (3) A process for preparing mesenchymal stem cells that express and secrete insulin in response to changes in glucose concentration, including transfection, virus packaging, transduction, cell screening, and detection methods for insulin expression and secretion.

[0019] The present invention is achieved through the following schemes.

[0020] The present invention provides a method for preparing mesenchymal stem cells that express insulin in response to changes in glucose, comprising the following steps:

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

[0022] S1.1. Determination of promoters of gene expression regulated in response to glucose concentration: By consulting literature, we found that the expression of genes such as pyruvate kinase L / R (PKLR), fatty acid synthase (FASNorFAS), acetyl-CoA carboxylase alpha (ACACA) and carbohydrate response element binding protein (CHREBP, also known as MLX interacting protein like: MLXIPL) was affected by glucose concentration or carbohydrate metabolism; then 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 use primers synthesized by a biological company to amplify the promoters of the above four genes by polymerase chain reaction (PCR); at the same time, culture HEK293T cells and extract genomic DNA;

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

[0025] The PCR reaction conditions were as follows: denaturation at 95°C for 5 min; then denaturation at 95°C for 1 min, annealing at 55°C for 20 sec, and extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min.

[0026] The PCR products were identified by agarose gel electrophoresis.

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

[0028] S1.4, screening promoters that respond to glucose to regulate reporter gene expression: First, determine that the lowest glucose concentration to maintain normal growth of HeLa cells is 0.3 mg / mL, which is sufficient to maintain normal growth of HeLa cells;

[0029] Then, the reporter gene expression vectors driven by the above promoters were transfected into HeLa cells cultured at different sugar concentrations. At the same time, a promoter that does not respond to glucose concentration to regulate gene expression (Adenovirus majorlate promoter: AdML) was used as a negative control. After 48 hours, the cells were collected, lysed, and the cell lysate was centrifuged. The supernatant was retained and used for luciferase activity detection.

[0030] Statistical analysis of the test data was then completed; the results showed that the FASN, ACACA and MLXIPL promoters increased the expression of the luciferase gene as the glucose concentration increased, while the PKLR promoter could not regulate the expression of luciferase in response to changes in glucose.

[0031] S2. Preparation of bone marrow mesenchymal stem cells that secrete mouse and human insulin in response to changes in glucose:

[0032] S2.1. Construction of a lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose changes: Using the recombination ligation method, the FASN, ACACA and MLXIPL promoters were used to replace the EF1α promoter on the pLV-EF1α-mCherry lentiviral vector to drive the expression of the downstream mCHerry-2A-hInsulin-His fusion gene. The PCR test results showed that the above promoters had replaced the EF1α promoter;

[0033] At the same time, mouse pancreatic tissue and PCR were used to amplify the mouse insulin gene and the obtained gene replaced hInsulin in the above mCherry-2A-hInsulin-His to form an mCherry-2A-mInsulin-His fusion gene and its expression was driven by a promoter that responds to changes in glucose; PCR detection results showed that mInsulin had replaced hInsulin.

[0034] S2.2. Transfection, screening and preparation of bone marrow mesenchymal stem cells that secrete mouse insulin in response to changes in glucose:

[0035] (a) The lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above that drive the expression of mouse insulin fusion gene in response to glucose changes are transfected into 293T cells to complete lentiviral packaging. After 72 hours, the cell culture fluid containing the lentivirus is collected and filtered through a 0.45 μm filter for later use;

[0036] (b) culturing mouse bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells;

[0037] (c) After 48 hours of infection, puromycin was used for selection for 48 hours, and then a small amount of cells were diluted in a 96-well plate to select monoclonal cells; the level of insulin expression by different promoters was determined by immunoblotting; the results showed that FASN and MXLIPL promoters were able to drive mouse insulin (mInsulin) expression significantly stronger than ACACA promoter;

[0038] (d) Insulin-expressing mesenchymal stem cells were cultured in a culture medium containing different concentrations of glucose. After 48 hours, the cells were collected and immunoblotting was performed to detect the expression of insulin in cells under different glucose concentrations. The results showed that the expression of mouse insulin driven by the FASN and MXLIPL promoters increased with the increase of glucose concentration, while the expression level of insulin driven by ACACA was low and its expression was not significantly affected by glucose concentration. By analyzing the secretion of insulin in the culture medium, the results showed that insulin was detected in the culture medium, indicating that the expressed insulin could be successfully secreted outside the cell.

[0039] S2.3. Transfection, screening and preparation of bone marrow mesenchymal stem cells secreting human insulin:

[0040] (a) Transfecting the lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above, which drive the expression of human insulin fusion gene in response to glucose changes, into 293T cells to complete lentiviral packaging, and collecting the cell culture fluid containing the lentivirus after 72 hours, filtering it through a 0.45 μm filter and setting it aside;

[0041] (b) culturing human bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing a lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells;

[0042] (c) After 48 hours of infection, puromycin was used for selection for 72 hours, and then a small amount of cells were diluted in a 96-well plate to select monoclonal cells. The level of insulin expression by different promoters was determined by immunoblotting; the results showed that the FASN and MXLIPL promoters were able to drive the expression of human insulin (hInsulin) significantly stronger than the ACACA promoter, and the latter drove a very low level of insulin expression;

[0043] (d) Mesenchymal stem cells with high insulin expression were cultured in culture medium containing different concentrations of glucose. After 48 hours, the cells were collected and immunoblotting analysis was performed to detect the expression of insulin in cells under different glucose concentrations. The results showed that the expression of human insulin driven by the FASN and MXLIPL promoters increased with the increase of glucose concentration. By analyzing the insulin secretion in the culture medium, the results showed that insulin was detected in the culture medium, indicating that the expressed insulin could be successfully secreted outside the cell.

[0044] Compared with the related art, the method for preparing mesenchymal stem cells expressing insulin in response to glucose changes provided by the present invention has the following beneficial effects:

[0045] The mesenchymal stem cells expressing insulin prepared by the present invention can overcome many problems existing in the current mesenchymal stem cell therapy. First, the problem that mesenchymal 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 changes in glucose, the stem cells prepared by the present invention can simulate the changes in blood sugar in humans and animals to express insulin, avoiding dangerous situations such as hypoglycemia coma that may be caused by continuous high secretion.

[0046] The present invention has the potential for clinical transformation. Once successfully applied in clinical practice, it can treat patients with type 1 diabetes and late-stage type 2 diabetes, which will not only bring economic benefits to the country and region, but also reduce the country's medical burden and bring new hope to diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the agarose gel electrophoresis results of the promoter PCR product in the present invention;

[0048] Figure 2 This is a diagram showing the result of enzyme digestion identification of the promoter cloned in the reporter gene expression vector in the present invention;

[0049] Figure 3 The figure is the result of the effect of glucose concentration on the expression of the reporter gene driven by the promoter in the present invention, wherein a, b, c, and d represent the effect of different glucose concentrations on the expression activity of luciferase driven by the PKLR, FASN, ACACA, and MXLIPL promoters, respectively, and ADML is an adenovirus promoter that does not contain a carbohydrate response element;

[0050] Figure 4 This is a diagram showing the electrophoresis results of the promoter cloned in the pLV-mCHerry-2A-hInsulin-His lentiviral expression vector for regulating gene expression in response to glucose concentration detected by PCR in the present invention;

[0051] Figure 5 This is the electrophoresis result diagram of the cloned mouse insulin gene (mInsulin, mINS) driven by different promoters detected by PCR in the present invention;

[0052] Figure 6The results of immunoblotting for the effects of different glucose concentrations on the expression of mouse insulin genes driven by different promoters in mouse bone marrow mesenchymal stem cells in the present invention are as follows: (a) immunoblotting to detect the expression of mouse insulin genes driven by different promoters in mouse bone marrow mesenchymal stem cells; (bd) immunoblotting to detect the expression of mouse insulin genes driven by FASN, ACACA, and MXLIPL promoters in mouse bone marrow mesenchymal stem cells at different glucose concentrations; (e) immunoblotting to analyze insulin secretion in cell culture medium;

[0053] Figure 7 The figures are the results of immunoblotting for the effect of different glucose concentrations on the expression of human insulin gene driven by different promoters in human bone marrow mesenchymal stem cells in the present invention, (a) immunoblotting detection of the expression of human insulin gene driven by different promoters in mouse bone marrow mesenchymal stem cells; (b and c) immunoblotting detection of the expression of mouse insulin gene driven by FASN and MXLIPL promoters in mouse bone marrow mesenchymal stem cells under different glucose concentrations; (d) immunoblotting analysis of the secretion of human insulin in cell culture medium. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0055] The present invention provides a method for preparing mesenchymal stem cells that express insulin in response to changes in glucose, comprising the following steps:

[0056] S1. Cloning and screening of promoters that can regulate gene expression in response to changes in glucose:

[0057] S1.1. Determination of promoters of gene expression regulated in response to glucose concentration: By consulting literature, we found that the expression of genes such as pyruvate kinase L / R (PKLR), fatty acid synthase (FASNorFAS), acetyl-CoA carboxylase alpha (ACACA) and carbohydrate response element binding protein (CHREBP, also known as MLX interacting protein like: MLXIPL) was affected by glucose concentration or carbohydrate metabolism; then the promoter sequences of these genes were downloaded from the eukaryotic promoter database.

[0058] S1.2. Primer design and PCR amplification of promoters: Design and use primers synthesized by a biological company to amplify the promoters of the above four genes by polymerase chain reaction (PCR); at the same time, culture HEK293T cells and extract genomic DNA;

[0059] The PCR reaction system included: 50 ng genomic DNA, 1.0 μL each of the forward and reverse primers for PCR amplification (25 μM), 25 μL of Phanata Max PCR 2 reaction mixture (Novagen), and water was added to make up to 50 μL;

[0060] The PCR reaction conditions were as follows: denaturation at 95°C for 5 min; then denaturation at 95°C for 1 min, annealing at 55°C for 20 sec, and extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min.

[0061] The PCR products were identified by agarose gel electrophoresis (see attached Figure 1 shown).

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

[0063] S1.4, screening promoters that respond to glucose to regulate reporter gene expression: First, determine that the lowest glucose concentration to maintain normal growth of HeLa cells is 0.3 mg / mL, 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 at different sugar concentrations. At the same time, a promoter that does not respond to glucose concentration to regulate gene expression (Adenovirus majorlate promoter: AdML) was used as a negative control. After 48 hours, the cells were collected, lysed, and the cell lysate was centrifuged. The supernatant was retained and used for luciferase activity detection.

[0065] The test data were then statistically analyzed; the results showed that the FASN, ACACA and MLXIPL promoters increased the expression of the luciferase gene with increasing glucose concentration (see Appendix Figure 3 ), while the PKLR promoter could not regulate the expression of luciferase in response to changes in glucose.

[0066] S2. Preparation of bone marrow mesenchymal stem cells that secrete mouse and human insulin in response to changes in glucose:

[0067] S2.1. Construction of a lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose changes: Using the recombination ligation method, the FASN, ACACA and MLXIPL promoters were used to replace the EF1α promoter on the pLV-EF1α-mCherry lentiviral vector to drive the expression of the downstream mCHerry-2A-hInsulin-His fusion gene. The PCR test results showed that the above promoters had replaced the EF1α promoter (see attached). Figure 4 shown);

[0068] At the same time, mouse pancreatic tissue and PCR were used to amplify the mouse insulin gene and the obtained gene was substituted for hInsulin in the above mCherry-2A-hInsulin-His to form the mCherry-2A-mInsulin-His fusion gene and driven by a promoter that responds to changes in glucose; PCR test results showed that mInsulin had replaced hInsulin (see attached Figure 5 shown).

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

[0070] (a) The lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above that drive the expression of mouse insulin fusion gene in response to glucose changes are transfected into 293T cells to complete lentiviral packaging. After 72 hours, the cell culture fluid containing the lentivirus is collected and filtered through a 0.45 μm filter for later use;

[0071] (b) culturing mouse bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells;

[0072] (c) After 48 h of infection, cells were screened with puromycin for 48 h, and then a small amount of cells were diluted in a 96-well plate to screen monoclonal cells. The levels of insulin expression by different promoters were determined by immunoblotting. The results showed that the FASN and MXLIPL promoters were able to drive mouse insulin (mInsulin) expression significantly stronger than the ACACA promoter (see attached). Figure 6 a);

[0073] (d) Insulin-expressing mesenchymal stem cells were cultured in medium containing different concentrations of glucose. After 48 h, the cells were collected and immunoblot analysis was performed to detect the expression of insulin in the cells under different glucose concentrations. The results showed that the expression of mouse insulin driven by the FASN and MXLIPL promoters increased with the increase of glucose concentration, while the expression level of insulin driven by ACACA was low and was not significantly affected by glucose concentration (see attached figure). Figure 6 bd). By analyzing the insulin secretion in the culture medium, the results showed that insulin was detected in the culture medium (see attached Figure 6 e), indicating that the expressed insulin can be successfully secreted outside the cell.

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

[0075] (a) Transfecting the lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above, which drive the expression of human insulin fusion gene in response to glucose changes, into 293T cells to complete lentiviral packaging, and collecting the cell culture fluid containing the lentivirus after 72 hours, filtering it through a 0.45 μm filter and setting it aside;

[0076] (b) culturing human bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing a lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells;

[0077] (c) After 48 hours of infection, puromycin was used for selection for 72 hours, and then a small amount of cells were diluted in a 96-well plate to select monoclonal cells. The level of insulin expression by different promoters was determined by immunoblotting; the results showed that the FASN and MXLIPL promoters were able to drive human insulin (hInsulin) expression significantly stronger than the ACACA promoter, and the latter drove a very low level of insulin expression (see Figure 2). Figure 7 a);

[0078] (d) Mesenchymal stem cells that highly express insulin were cultured in medium containing different concentrations of glucose. After 48 h, the cells were collected and immunoblot analysis was performed to detect the expression of insulin in cells under different glucose concentrations. The results showed that the expression of human insulin driven by the FASN and MXLIPL promoters increased with increasing glucose concentrations (see Figure 2A). Figure 7 b and 7c). By analyzing the insulin secretion in the culture medium, the results showed that insulin was detected in the culture medium (as shown in the attached Figure 7 d), indicating that the expressed insulin can be successfully secreted outside the cell.

[0079] It should be noted that, since mesenchymal stem cells may 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 mesenchymal stem cells, muscle mesenchymal stem cells, but they all have the characteristics of mesenchymal stem cells and can differentiate into osteoblasts, chondrocytes, adipocytes, etc. Although the present invention uses human and mouse bone marrow mesenchymal stem cells, it may also have transformation potential using other mesenchymal stem cells and the technology of the present invention. Therefore, the use of mesenchymal stem cells from other tissue sources and the technology of the present invention to prepare mesenchymal stem cells that express and secrete insulin is also within the scope of protection of the present invention.

[0080] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing mesenchymal stem cells that express insulin in response to changes in glucose, characterized in that: The following steps are involved: S1. Cloning and screening of promoters that can regulate gene expression in response to changes in glucose; S1.1, identification of promoters that regulate gene expression in response to glucose concentration; S1.2, primer design and PCR amplification of promoter; S1.3, promoter-driven reporter gene cloning; S1.4, screening for 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 changes in glucose: S2.

1. Constructing a lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to changes in glucose; S2.2, transfection, screening and preparation of bone marrow mesenchymal stem cells that secrete mouse insulin in response to changes in glucose; S2.

3. Transfection, screening and preparation of bone marrow mesenchymal stem cells that secrete human insulin.

2. The method for preparing mesenchymal stem cells expressing insulin in response to glucose changes according to claim 1, characterized in that: The specific steps of determining the promoter of gene expression regulated in response to glucose concentration in step S1.1 are as follows: By consulting the literature, we found that the expression of 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 proteinlike: MLXIPL) genes was affected by glucose concentration or carbohydrate metabolism; then the promoter sequences of these genes were downloaded from the eukaryotic promoter database.

3. The method for preparing mesenchymal stem cells expressing insulin in response to glucose changes according to claim 1, characterized in that: The specific steps of primer design and PCR amplification of promoter in step S1.2 are: Primers were designed and synthesized by a biological company for polymerase chain reaction (PCR) amplification of the promoters of the above four genes; at the same time, HEK293T cells were cultured and used for genomic DNA extraction; The PCR reaction system included: 50 ng genomic DNA, 1.0 μL each of the forward and reverse primers for PCR amplification (25 μM), 25 μL of Phanata Max PCR 2 reaction mixture (Novagen), and water was added to make up to 50 μL; The PCR reaction conditions were as follows: denaturation at 95°C for 5 min; then denaturation at 95°C for 1 min, annealing at 55°C for 20 sec, and extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min. The PCR products were identified by agarose gel electrophoresis.

4. The method for preparing mesenchymal stem cells expressing insulin in response to glucose changes according to claim 1, characterized in that: The specific steps of promoter-driven reporter gene cloning in step S1.3 are as follows: The PKLR, FASN, ACACA and MLXIPL promoters were loaded into the reporter gene expression vector pGL3-Basic to drive the expression of the downstream luciferase gene.

5. The method for preparing mesenchymal stem cells expressing insulin in response to glucose changes according to claim 1, characterized in that: The specific steps of screening the promoter that regulates the expression of the reporter gene in response to glucose in step S1.4 are: First, it was determined that the lowest glucose concentration to maintain normal growth of HeLa cells was 0.3 mg / mL, which was sufficient to maintain normal growth of HeLa cells; Then, the reporter gene expression vectors driven by the above promoters were transfected into HeLa cells cultured at different sugar concentrations. At the same time, a promoter that does not respond to glucose concentration to regulate gene expression (Adenovirus major late promoter: AdML) was used as a negative control. After 48 hours, the cells were collected, lysed, and the cell lysate was centrifuged. The supernatant was retained and used for luciferase activity detection. Then statistical analysis of the test data was completed.

6. The method for preparing mesenchymal stem cells expressing insulin in response to glucose changes according to claim 1, characterized in that: The specific steps of constructing a lentiviral vector expressing human insulin (hInsulin) or mouse insulin (mInsulin) fusion gene in response to glucose changes in step S2.1 are: By using the recombination ligation method, the FASN, ACACA and MLXIPL promoters were used to replace the EF1α promoter on the pLV-EF1α-mCherry lentiviral vector to drive the expression of the downstream mCHerry-2A-hInsulin-His fusion gene; At the same time, mouse pancreatic tissue and PCR were used to amplify the mouse insulin gene, and the obtained gene replaced the hInsulin in the above mCherry-2A-hInsulin-His to form the mCherry-2A-mInsulin-His fusion gene and was expressed by a promoter that responded to changes in glucose.

7. The method for preparing mesenchymal stem cells expressing insulin in response to changes in glucose according to claim 6, characterized in that: The specific steps of transfecting, screening and preparing bone marrow mesenchymal stem cells that secrete mouse insulin in response to changes in glucose in step S2.2 are: (a) The lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above that drive the expression of mouse insulin fusion gene in response to glucose changes are transfected into 293T cells to complete lentiviral packaging. After 72 hours, the cell culture fluid containing the lentivirus is collected and filtered through a 0.45 μm filter for later use; (b) culturing mouse bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells; (c) After 48 h of infection, cells were screened with puromycin for 48 h, and then a small amount of cells were diluted in a 96-well plate to screen monoclonal cells; the levels of insulin expression by different promoters were determined by immunoblotting; (d) Insulin-expressing mesenchymal stem cells were cultured in culture medium containing different concentrations of glucose. After 48 h, the cells were collected and immunoblot analysis was performed to detect the expression of insulin in the cells under different glucose concentrations.

8. The method for preparing mesenchymal stem cells expressing insulin in response to changes in glucose according to claim 6, characterized in that: The specific steps of transfecting, screening and preparing bone marrow mesenchymal stem cells that secrete human insulin in step S2.3 are: (a) Transfecting the lentiviral vector and packaging vectors pH1 and pH2 constructed in step S2.1 above, which drive the expression of human insulin fusion gene in response to glucose changes, into 293T cells to complete lentiviral packaging, and collecting the cell culture fluid containing the lentivirus after 72 hours, filtering it through a 0.45 μm filter and setting it aside; (b) culturing human bone marrow mesenchymal stem cells in a 12-well plate, and when the cells grow to 60% confluence, culturing the mesenchymal stem cells with a culture medium containing a lentivirus so that the lentivirus can infect the bone marrow mesenchymal stem cells; (c) After 48 h of infection, puromycin was used for selection for 72 h, and then a small amount of cells were diluted in a 96-well plate to select monoclonal cells. Immunoblotting was used to determine the level of insulin expression from different promoters; (d) Mesenchymal stem cells that highly express insulin were cultured in culture medium containing different concentrations of glucose. After 48 hours, the cells were collected and immunoblot analysis was performed to detect the expression of insulin in the cells under different glucose concentrations.

Images