Use of iPSC-induced differentiation into pancreatic progenitor cells combined with medication to treat diabetes

Abstract

The present invention relates to the biological field. Further, the present invention provides the use of iPSC induced differentiation into pancreatic progenitor cells combined with medication for the treatment of diabetes. Specifically, the present invention provides a monoclonal antibody that specifically inhibits the activity of BMP4, which can inhibit the differentiation of iPSC cells into common osteoblasts and adipocytes by inhibiting the activity of BMP4 protein, thereby promoting the transformation of iPSC into pancreatic progenitor cells and differentiation into islet-like cells. After the islet-like cells and ginsenoside Rb3 are combined to prepare a drug kit, the effect of lowering blood sugar can be significantly improved, and it has a good prospect for drug application.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to the use of iPSCs induced to differentiate into pancreatic progenitor cells combined with medication to treat diabetes. Background Art

[0002] At present, due to many bad eating and living habits of people, diabetic patients are showing a "younger age" trend. According to the World Health Organization (WHO) Global Diabetes Report, the number of diabetic patients reached 536.6 million in 2021, and is expected to increase to 783.2 million in 2045, which means that more than 10.5% of the world's population suffers from diabetes.

[0003] There is currently no treatment that can cure diabetes. Domestic treatment methods mainly control blood sugar levels as close to normal as possible to delay or prevent the occurrence of further diabetic complications. Conventional treatment options generally include oral hypoglycemic drugs and insulin injections. In addition, physical exercise and a healthy diet also play a key role in disease control. Although modern insulin and drug therapies are constantly improving, exogenous insulin can never be as accurate and dynamic as endogenous beta cell insulin secretion. Long-term insulin injections can also lead to hypoglycemia, insulin resistance, weight gain and other problems.

[0004] Stem cells are cells with the potential for self-renewal and multidirectional differentiation. Under certain conditions, they can differentiate into a variety of functional cells and have the potential to regenerate various tissues and human organs. By manipulating the fate of stem cell differentiation through technical means, so that they can differentiate in the direction we need, we can provide new treatment strategies for difficult diseases. However, due to the ethical and moral debates about the source of stem cells, the development of new sources of stem cells is a key research direction.

[0005] Since iPSC cells can be induced from autologous cells, there is no immune rejection in the treatment of diseases, so iPSC cells have a wider application than ES cells. They have great application value in regenerative medicine, clinical medicine, tissue engineering and pharmacology, and have become an important research direction in the field of life medicine. Exogenous reprogramming factors are expressed in somatic cells from patients to induce the production of iPSC cells, and defective genes are corrected through genetic modification, and then differentiated to obtain the required cells with normal functions for cell transplantation therapy, such as differentiation to produce motor neurons with normal functions to treat amyotrophic lateral sclerosis.

[0006] The latest research shows that iPSC can not only generate specific cell types, but also has important significance in disease treatment, drug screening and regenerative medicine. iPSC treatment of T1DM belongs to the category of regenerative medicine. Its basic principle is to reverse somatic cells into early undifferentiated pluripotent stem cells, and then treat them with various cytokines to mimic the growth and development process of embryonic cells, inducing them to become pancreatic β cells with insulin secretion function. Some scholars used 4 viral transcription factors to reprogram normal mouse skin fibroblasts into iPSC, and then induced iPSC to differentiate into pancreatic β-like cells in vitro, and transplanted them into diabetic mice by portal vein injection. The fasting blood sugar of mice was monitored 2 days after surgery, and it was found that the blood sugar levels of transplanted mice were normal, while the mice in the control group were still in a state of hyperglycemia. Other studies have shown that iPSCs were derived from NOD / SCID mouse fibroblasts and induced into functional islet cells using a directed differentiation method. STZ was then used to induce NOD / SCID mice to establish a diabetes model, and functional islet cells were transplanted under the renal capsule of the mice. It was found that the blood glucose levels of the mice gradually returned to normal 2-4 days after transplantation, while the blood glucose of the control group mice was >18.3mmol / L. In addition, mouse embryonic fibroblasts were reprogrammed into iPSCs by lentiviral transfection, and iPSCs were induced to differentiate into islet-like cells. The islet-like cells were then transplanted into the left lobe of the liver of STZ-induced diabetic mice. After 6-7 days, the insulin secretion and blood glucose levels of the mice gradually normalized, indicating that iPSC-derived islet-like cells have the ability to reverse hyperglycemia. In addition, human iPSCs were induced to differentiate into pancreatic β cells, and then STZ-induced SCID mice were used to establish a diabetic model. The pancreatic β cells were transplanted into the left renal capsule of SCID diabetic mice. The control group was injected with phosphate-buffered saline (PBS), and blood was collected from the tail vein once a week to monitor the blood glucose levels of the mice. It was found that among the 20 diabetic mice transplanted with pancreatic β cells, the blood glucose levels of 6 mice gradually decreased within 4 weeks after surgery, while the mice in the control group still maintained a high blood glucose level.

[0007] It can be seen from the research of existing technologies that iPSC cells are already important research cells for the treatment of diabetes. However, at present, there are not enough studies on the induction and differentiation of iPSC cells into pancreatic progenitor cells, and the differentiation efficiency is still relatively low. It is necessary to develop new and efficient differentiation methods to improve the harvesting efficiency of pancreatic progenitor cells, which is conducive to promoting the research of clinical treatment of insulin. The inventor has previously studied the method of inducing iPSC to differentiate into pancreatic progenitor cells to repair the pancreas. On this basis, the development of corresponding pancreatic progenitor cell combination drugs for the treatment of diabetes is an important research direction. Summary of the invention

[0008] The present invention overcomes the defects of the prior art and, based on previous studies, provides a method for treating diabetes by inducing iPSCs to differentiate into pancreatic progenitor cells in combination with medication.

[0009] Furthermore, the present invention provides a medicine kit for treating diabetes, wherein the medicine kit comprises islet-like cells differentiated from pancreatic progenitor cells and ginsenoside Rb3.

[0010] Specifically, the pancreatic progenitor cells are prepared by the method of the present invention for improving the induction of differentiation of iPSC cells into pancreatic progenitor cells. Further, the specific method is disclosed in the applicant's previous patent application (CN2024107981505).

[0011] The pancreatic progenitor cells were further induced for 11 days in a DMEM high-glucose medium containing 1% ITS, 10 ng / ml HGF, 20 mmol / L nicotinamide, 0.1 mmol / L β-mercaptoethanol, 10 μmol / L LY294002, and 30 μmol / L B27, and relatively regular round islet-like cells were harvested.

[0012] Specifically, the dosage of ginsenoside Rb3 is 30 mg / kg, and the number of islet-like cells is 1×10 6 Pieces / times.

[0013] Furthermore, the present invention also provides a method for improving the induction of differentiation of iPSC cells into pancreatic progenitor cells.

[0014] Specifically, the method includes using a BMP4 protein inhibitor on iPSC cells.

[0015] Specifically, the inhibitor inhibits the differentiation of iPSC cells into osteoblasts and adipocytes by inhibiting the action of BMP4 protein, thereby promoting the transformation of iPSC cells into pancreatic progenitor cells.

[0016] Specifically, the BMP4 protein inhibitor is a monoclonal antibody specific to BMP4.

[0017] Specifically, the monoclonal antibody specific for BMP4 is B2D5, the light chain variable region sequence of which is shown in SEQ ID NO:1, and the heavy chain variable region sequence of which is shown in SEQ ID NO:2.

[0018] Furthermore, the antibodies of the present invention may also be variants having one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3.

[0019] The present invention further provides a vector comprising at least one of the above-mentioned nucleic acids.

[0020] Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which is capable of encoding the amino acid.

[0021] Further, it is also intended to provide antibody coding regions for the present invention by using standard molecular biology techniques to change existing antibody genes, and obtain variants (agonists) of antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions and substitutions in the amino acid sequence of anti-BMP4 antibodies or peptides. For example, one type of substitution is a conservative amino acid substitution. Such substitutions are substitutions of a given amino acid in an anti-BMP4 antibody with another amino acid of similar nature. It is generally considered to be a conservative substitution: substitutions between aliphatic amino acids Ala, Val, Leu and Ile; interchanges of hydroxyl residues Ser and Thr; interchanges of acidic residues Asp and Glu; exchanges between amide residues Asn and Gln; exchanges of basic residues Lys and Arg; substitutions between aromatic residues Phe, Tyr, and the like. Further, variants or agonists or peptides of anti-BMP4 antibodies may be fully functional, or may lack one or more active functions. Fully functional variants generally contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants may also contain similar amino acid substitutions, which do not result in functional changes or insignificant changes. Alternatively, such substitutions may positively or negatively affect function to some extent. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions or truncations, or substitutions, insertions, inversions or deletions in key residues or key regions.

[0022] The amino acid that is essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. The latter method introduces a single alanine mutation at each residue in the molecule. The biological activity of the mutant molecule obtained is then tested, such as epitope binding or in vitro ADCC activity. It is also possible to determine the site that is crucial for ligand-receptor binding by structural analysis (such as crystallography, nuclear magnetic resonance or photoaffinity labeling). In addition, antibodies can also often contain amino acids other than twenty kinds of "naturally occurring" amino acids. In addition, many amino acids (including terminal amino acids) can be modified by natural methods (such as processing or other post-translational modifications) or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, formation of disulfide bonds, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, formation of a GPI anchor, hydroxylation, iodination, methylation, myristoylation, oxidation, hydrolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, t-RNA-mediated addition of amino acids to proteins (e.g., arginylation and ubiquitination).

[0023] Furthermore, the present invention also provides a method for improving the induction differentiation of iPSC cells into pancreatic progenitor cells, the method comprising using a corresponding induction medium and an induction method, wherein the induction medium comprises a monoclonal antibody B2D5 against BMP4.

[0024] Furthermore, the method of inducing iPSC cells to differentiate into pancreatic progenitor cells of the present invention specifically comprises using 200U / mL collagenase IV to digest iPSC clusters into relatively loose small cell clusters, placing them in a culture plate coated with Matrigel, and culturing them for 4 days in DMEM / F12 containing 100ng / mL activin A, 1M wortmannin, 10μM Y-27632, and 100μg / mL BMP4 monoclonal antibody B2D5 when 60% fusion is reached; then the cultured cells are continuously placed in F12 / IMDM containing 2μM RA (retinoic acid), 20ng / ml bFCF (basic fibroblast growth factor) and 50 ng / mL NOGGIN, 10μM Y-27632, and 100μg / mL BMP4 monoclonal antibody B2D5 for 4 days; and then 50ng / mL EGF, 10μM Pancreatic progenitor cells were expanded and differentiated after 5 days of culture in F12 / IMDM with Y-27632 and 100 μg / mL BMP4 monoclonal antibody B2D5.

[0025] The present invention further provides the use of islet-like cells and ginsenoside Rb3 in preparing a drug kit for treating diabetes, wherein the islet-like cells are prepared by a method for improving the induction and differentiation of iPSC cells into islet-like cells, the method comprising the step of inducing iPSC using an induction medium containing the monoclonal antibody B2D5 of BMP4; the induction step specifically comprises using 200U / mL collagenase IV to digest the iPSC clusters into relatively loose small cell clusters, placing them in a culture plate coated with Matrigel, and culturing them for 4 days in DMEM / F12 containing 100ng / mL activin A, 1M wortmannin, 10μM Y-27632, and 100μg / mL BMP4 monoclonal antibody B2D5 when 60% fusion is reached; then the cultured cells are continuously placed in a medium containing 2μM RA, 20ng / ml bFCF and 50 ng / mL NOGGIN, 10μM Y-27632, 100 μg / mL BMP4 monoclonal antibody B2D5 in F12 / IMDM and cultured for 4 days; then placed in 50 ng / mL EGF, 10 μM Y-27632, 100 μg / mL BMP4 monoclonal antibody B2D5 in F12 / IMDM and cultured for 5 days to expand and differentiate pancreatic progenitor cells; the pancreatic progenitor cells were further induced for 11 days in DMEM high-glucose culture medium containing 1% ITS, 10 ng / ml HGF, 20 mmol / L nicotinamide, 0.1 mmol / L β-mercaptoethanol, 10 μmol / L LY294002, and 30 μmol / L B27 to obtain relatively regular round islet-like cells.

[0026] Beneficial effects: The present invention provides the use of iPSC induced differentiation into pancreatic progenitor cells combined with medication for the treatment of diabetes. Specifically, the present invention provides a monoclonal antibody that specifically inhibits the activity of BMP4, which can inhibit the differentiation of iPSC cells into common osteoblasts and adipocytes by inhibiting the activity of BMP4 protein, thereby promoting the transformation of iPSC into pancreatic progenitor cells and differentiation into islet-like cells. After the islet-like cells and ginsenoside Rb3 are combined to prepare a drug kit, the effect of lowering blood sugar can be significantly improved, and it has a good prospect for drug application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of the effect of BMP4 monoclonal antibody B2D5 on BMP4 activity

[0028] Figure 2 The results of the expression changes of characteristic genes of pancreatic progenitor cells are shown in Figure 2

[0029] Figure 3 The results of each group on the fasting blood glucose of rats DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0031] Example 1 Development of BMP4 monoclonal antibody

[0032] Recombinant human BMP4 protein (ab238298, abcam) was selected as the immunogen. Balb / c mice were used as immunized mice. Balb / c mice and mouse myeloma cell lines (SP2 / 0) were purchased from the Chinese Animal Health and Epidemiology Center.

[0033] The first immunization was performed with Freund's complete adjuvant emulsified BMP4 protein, and subcutaneously injected into 8-week-old Balb / c mice (90 μg / mouse); the second and third immunizations were performed with Freund's incomplete adjuvant emulsified recombinant protein, and subcutaneously injected into immunization (90 μg / mouse); the interval between each immunization was 14 days. A booster immunization was performed once with a dose of 50 μg / mouse intraperitoneally 3 days before fusion. Three days after the booster immunization of Balb / c mice, the mouse spleen cells were aseptically obtained and fused with SP2 / 0 myeloma cells under the action of PEG1500 according to the conventional method. The fused cells were selectively cultured in HAT medium containing 200 mL / L fetal bovine serum, and the medium was half changed after 5 days. After 10 days, HT medium was used for culture. When the cells grew to more than 2 / 5 of the culture wells, the culture supernatant was aspirated to screen the positive wells by indirect ELISA method, and the positive hybridoma cells were cloned three times by limiting dilution method until the positive rate of the clone wells reached 100%. After expansion and culture, a hybridoma cell line B2D5 that stably secreted antibodies was obtained after screening.

[0034] Positive hybridoma cells B2D5 were cultured at 8×10 5The dose of 10000 / mouse was intraperitoneally injected into Balb / c mice sensitized with liquid paraffin. After 10 days, the ascites was collected. It can be taken multiple times, centrifuged at 10000r / min for 10min, and the supernatant was collected, labeled and frozen. Enzyme-linked immunosorbent assay The purified recombinant BMP4 protein and SP2 / 0 myeloma cell protein lysate were diluted to 2μg / mL with coating solution, and coated on ELISA plates respectively. The prepared monoclonal antibody ascites and hybridoma culture supernatant were used as primary antibodies, and HRP-labeled goat anti-mouse IgG was used as secondary antibody for indirect ELISA. The results are shown in Table 1.

[0035] Table 1 Specificity analysis of monoclonal antibody B2D5

[0036] antigen Hybridoma cell culture supernatant ascites Recombinant BMP4 protein + + SP2 / 0 Myeloma Cell Lysate - -

[0037] As can be seen from Table 1, both the hybridoma cell culture supernatant and ascites can specifically react with the recombinant BMP4 protein, but not with the SP2 / 0 myeloma cell protein lysate, showing good specificity.

[0038] The hybridoma cell was subjected to variable region identification, and the light chain variable region sequence thereof was shown as SEQ ID NO:1, and the heavy chain variable region sequence thereof was shown as SEQ ID NO:2.

[0039] The monoclonal antibody is purified by column and then used for later use.

[0040] Example 2 Affinity identification of BMP4 monoclonal antibody B2D5

[0041] The antibody affinity was determined using the Biacore 3000 system. 10mM NaAC at pH 4.0, pH 4.5, pH 5.0, and pH 5.5 was prepared, and the antibody to be tested was diluted by appropriate multiples. It was pre-concentrated on the CM5 chip, and the optimal NaAC at pH 4.5 was selected as the coating diluent. Coating: Dilute the antibody to be tested with NaAC at the optimal pH 4.5, select the optimal dilution and select a channel on the CM5 chip for coupling. The target value of the coupled antibody was 2500RU, and another channel was selected as a control. The experimental conditions were 25°C, a flow rate of 20μL / min, and the buffer was HBS-EP (pH 7.4). After the chip-coupled antibody reached the target value, the chip surface was closed. Regeneration condition analysis: 100nM BMP4-Fc flows through the chip surface to bind to the antibody on the chip surface. After stabilization, 10mM glycine-hydrochloric acid at pH3.5, pH2.5, pH2.0, pH1.5 and borate buffer at pH8.5 flow through the chip surface in sequence until the best regeneration effect is obtained to determine the best regeneration condition. Kinetic analysis of antibody-antigen binding: The concentration of antigen BMP4-Fc is determined by Broford method, and then the antigen sample is diluted with HBS-EP buffer. Different concentrations (5 different concentrations from 0-100nM) of BMP4-Fc flow through the test antibody channel and the control antibody channel, with a flow rate of 20μL / min, a binding time of 3min, a stabilization time of 1min, and a dissociation time of 15min. The regeneration conditions for each cycle are 10mM glycine-hydrochloric acid pH1.5 and borate buffer at pH8.5, a flow rate of 20μL / min, and each solution is regenerated for 30s. Affinity constant calculation: The obtained sensorgram was fitted with a 1:1 Langmuir binding mode using Bia-evaluation analysis software 4 to calculate the kinetic constants of antigen-antibody binding. The results showed that the affinity KD of the antibody was 6.39E-10 (mol / L), with Ka=5.13×10 4 [1 / (mol*s / L)] ,Kd=3.28×10 -5 (1 / s).

[0042] Example 3 Activity verification of BMP4 monoclonal antibody B2D5

[0043] iPSC cells (Cat. No. XG-X3374, Shanghai Sigma) were cultured. When the iPSC confluence reached 70%, the supernatant was removed and the cells were washed twice with preheated DPBS (Dulcerative Phosphate Buffer), and then preheated Tryple (recombinant enzyme tryple) was added to digest the cells to a single cell state. After terminating the digestion and centrifuging, the supernatant was removed, and the cells were resuspended in complete culture medium and counted. The cell density was adjusted to 1×10 4 / mL, inoculate the same number of cells into six-well plates containing BMP4 monoclonal antibody B2D5 with final concentrations of 1μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively, and culture the cells in a 5% CO2, 37°C constant temperature incubator for 24 hours. LDN-212854 was used as a positive control at a concentration of 100μg / mL. Take 1ml of cells from each group at 3000r / min, wash with PBS three times, and then use an equal amount of PBS to break and extract bacterial proteins. Perform SDS-PAGE electrophoresis and transfer to PVDF membrane. Use the prepared monoclonal antibody as the primary antibody, HRP-labeled goat anti-mouse IgG as the secondary antibody, and DAB for color development. Using the blank group of cells without monoclonal antibody as the control, calculate the relative expression of BMP4 protein in each group. The results are as follows Figure 1 shown.

[0044] from Figure 1 It can be seen that with the increase of the concentration of BMP4 monoclonal antibody B2D5, the relative expression of BMP4 protein was gradually inhibited. When the highest concentration was 200 μg / mL, the relative expression of BMP4 protein was (0.05±0.02), and the inhibitory effect was better than that of the positive control group, indicating that the monoclonal antibody of the present invention has good biological activity.

[0045] The invention further detected the effect of the monoclonal antibody on cell activity and found that at 100 μg / mL, the BMP4 protein inhibitory activity and apoptosis effect on iPSC cells were in the best balance state, and this concentration was selected for subsequent experiments.

[0046] Example 4 Inducing differentiation of iPSC cells into pancreatic progenitor cells

[0047] Experimental group: iPSC cells (Cat. No. XG-X3374, Shanghai Sigma) were induced to differentiate. 200U / mL collagenase IV was used to digest iPSC clusters into loose small cell clusters, which were placed in a culture plate coated with Matrigel. When 60% confluence was achieved, the cells were cultured in DMEM / F12 containing 100ng / mL activin A, 1M wortmannin, 10μM Y-27632, and 100μg / mL BMP4 monoclonal antibody B2D5 for 4 days; the cultured cells were then placed in F12 / IMDM containing 2μM RA, 20ng / ml bFCF and 50 ng / mL NOGGIN, 10μM Y-27632, and 100μg / mL BMP4 monoclonal antibody B2D5 for 4 days; 50ng / mL EGF, 10μM Pancreatic progenitor cells were expanded and differentiated after 5 days of culture in F12 / IMDM with Y-27632 and 100 μg / mL BMP4 monoclonal antibody B2D5.

[0048] In the control group, iPSC cells (Cat. No. XG-X3374, Shanghai Sigma) were induced to differentiate. 200U / mL collagenase IV was used to digest the iPSC clusters into loose small cell clusters, which were placed in a culture plate coated with Matrigel. When the confluence reached 60%, the cells were cultured in DMEM / F12 containing 100ng / mL activin A, 1M wortmannin, and 10μM Y-27632 for 4 days; then the cultured cells were continued to be cultured in F12 / IMDM containing 2μM RA, 20ng / ml bFCF and 50ng / mL NOGGIN, 10μM Y-27632 for 4 days; and then the cells were cultured in F12 / IMDM containing 50ng / mL EGF and 10μM Y-27632 for 5 days to expand and differentiate the pancreatic progenitor cells.

[0049] In the blank group, iPSC cells (Cat. No. XG-X3374, Shanghai Sigma) were cultured in F12 / IMDM medium for the same time as the control group without adding any induction factors.

[0050] The mRNA was extracted from the pancreatic progenitor cells obtained by the two methods and the iPSC cells in the blank group, and cDNA was obtained by reverse transcription with random primers in vitro. The relative expression levels of pancreatic progenitor cell characteristic genes in different samples were obtained using the SYBR Green dye method. -△△Ct Algorithm, calculate and statistically analyze the difference in expression level of target gene mRNA relative to internal reference GAPDH gene, and the primer sequences are shown below.

[0051] Ngn3: 5'-AGAGCGAGTTGGCACTGAGC / GTCCAGTGCCGAGTTGAGGT-3'

[0052] Nkx6.1:5'-ACACGAGACCCACTTTTTCCG / GGAACCAGACCTTGACCTGACTC-3'

[0053] Insulin: 5'-ACGAGGCTTCTTCTACACACC / TCCACAATGCCACGCTTCTG-3'

[0054] PDX-1: 5'-GTTGAACTTGACCGAGAGACACA / CTTCTTGTCCTCTCCTTTTTCC-3'

[0055] GAPDH: 5'-GTGGACCTGACCTGCCGTCI / GGAGGAGTGCGTGTCGCTGT-3'

[0056] Specific results such as Figure 2 shown.

[0057] The results of detecting changes in the expression of characteristic genes of pancreatic progenitor cells are as follows Figure 2 As shown, the expression of pancreatic progenitor cell-specific genes Ngn3, PDX-1, Nkx6.1 and pancreatic β cell marker gene Insulin in the experimental group increased significantly relative to the expression of GAPDH compared with the blank group (P<0.01), and the results of the experimental group were also significantly improved compared with the control group, which also shows that the experimental group of the present invention has a better effect of differentiation into pancreatic progenitor cells. This also shows that the BMP4 monoclonal antibody of the present invention can inhibit the differentiation of iPSC cells into common osteoblasts and adipocytes by inhibiting the activity of BMP4 protein, thereby promoting the transformation of iPSC into pancreatic progenitor cells.

[0058] Example 5 Differentiation of pancreatic progenitor cells into islet-like cells

[0059] The pancreatic progenitor cells obtained from the experimental group of Example 4 were induced for 11 days in a DMEM high-glucose medium containing 1% ITS, 10 ng / ml HGF, 20 mmol / L nicotinamide, 0.1 mmol / L β-mercaptoethanol, 10 μmol / L LY294002, and 30 μmol / L B27, and relatively regular round islet-like cells were harvested.

[0060] The pancreatic progenitor cells obtained from the control group of Example 4 were induced for 14 days in a DMEM high-glucose medium containing 1% ITS, 10 ng / ml HGF, 20 mmol / L nicotinamide, 0.1 mmol / L β-mercaptoethanol, 10 μmol / L LY294002, and 30 μmol / L B27, and then relatively regular round islet-like cells were harvested as controls.

[0061] The islet-like cells prepared by the above 2 methods were subjected to the expression of insulin and C-peptide positive cells. Specifically, the induced cells were made into a single cell suspension, fixed with 4% paraformaldehyde solution for 15 minutes, washed with PBS, treated with 0.3% Triton X-100, blocked with 0.5% bovine serum albumin, incubated with primary antibody, FITC-labeled fluorescent secondary antibody, incubated at room temperature in the dark for 30 minutes, washed with PBS and resuspended, and the expression of insulin and C-peptide positive cells was detected by flow cytometry. The results are as shown in the company's previous patent (CN2024107981505). The specific results are shown in Table 2.

[0062] Table 2 Analysis of islet-like cells in each group

[0063] Group Insulin-positive cells C-peptide positive cells Islet-like cells obtained from the experimental group （56.8±3.4）% （16.8±1.1）% Islet-like cells obtained from the control group （38.7±2.1）% （10.8±0.5）%

[0064] It can be seen from Table 2 that the islet-like cells prepared by the present invention have good islet cell characteristics.

[0065] Example 6 Islet-like cells combined with hypoglycemic experiment

[0066] Establishment of diabetic animal model: After 3 days of adaptive feeding, 10 rats were randomly selected as normal control group, and the rest were established into diabetic model. First, streptozotocin was prepared into 0.1% injection solution with 0.1 mol / L citric acid-sodium citrate buffer (pH 4.5), and ice bath was placed in the dark. After 18 hours of fasting, 70 mg / kg was intraperitoneally injected into rats. Blood sugar was tested 3 days after injection. If the blood sugar concentration was higher than 16.7 mol / L for 3 consecutive times, it indicated that the model was successfully established.

[0067] Animal grouping: After modeling, the rats were randomly divided into model control group, normal control group, islet-like cell treatment group obtained from the experimental group, islet-like cell treatment group obtained from the control group, positive control group (ginsenoside Rb3), and islet-like cell treatment group obtained from the experimental group combined with ginsenoside Rb3, with 10 rats in each group.

[0068] Ten days after modeling, the islet-like cells obtained from the experimental group (group A), the islet-like cells obtained from the control group (group B), and the islet-like cells obtained from the experimental group combined with ginsenoside Rb3 treatment group (group C) were transplanted with 1 mL of the corresponding cell suspension per mouse (cell number 1×10 6 The normal control group (Group D) and the model control group (Group E) were injected with 2 mL of culture medium without any cells through the tail vein. The transplantation was performed once every 2 weeks, for a total of 2 transplantations. The positive control group (Group F) was intragastrically administered with 30 mg / kg ginsenoside Rb3 once a day at the same time in the morning for 28 consecutive days; the islet-like cells obtained from the experimental group and the ginsenoside Rb3 treatment group were intragastrically administered with 30 mg / kg ginsenoside Rb3 once a day at the same time in the morning after the islet-like cells obtained from the experimental group were injected into the tail vein, for 28 consecutive days; 4 weeks after the second transplantation, all rats were killed and specimens were collected. The blood glucose levels of rats in each group were tested. When testing blood glucose, the tail end of the rat was first disinfected with 75% ethanol by volume, and then the fasting blood glucose level of the rat was tested by collecting blood from the tail vein using a Roche blood glucose meter. The results are shown in Figure 3 shown.

[0069] from Figure 3It can be seen that the blood glucose of the islet-like cell treatment group obtained in the experimental group, the islet-like cell treatment group obtained in the control group, the positive control group, and the islet-like cell combined with ginsenoside Rb3 treatment group obtained in the experimental group was significantly reduced compared with the model control group, and the difference was significant (P<0.01). The blood glucose value of the islet-like cell combined with ginsenoside Rb3 treatment group (group C) obtained in the experimental group was (5.32±0.41) mmol / L, which was basically close to the normal control group (group D). The blood glucose value of the islet-like cell treatment group (group A) obtained in the experimental group was also lower than that of the islet-like cell treatment group (group B) obtained in the control group, which shows that inhibiting the activity of BMP4 protein, inhibiting the differentiation of iPSC cells into common osteoblasts and adipocytes, and promoting the transformation of iPSC into pancreatic progenitor cells is more effective, the cell activity is higher, and the ability to produce insulin is stronger, thereby having a better ability to lower blood glucose.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a combination of islet-like cells and ginsenoside Rb3 in preparing a drug kit for treating diabetes, characterized in that: The islet-like cells are prepared by a method for improving the induction and differentiation of iPSC cells into islet-like cells. The method includes the step of inducing iPSC using an induction medium containing a monoclonal antibody B2D5 targeting BMP4, wherein the light chain variable region sequence of the monoclonal antibody B2D5 is shown in SEQ ID NO: 1, and the heavy chain variable region sequence thereof is shown in SEQ ID NO:

2. The step of inducing iPSC using an induction medium containing a monoclonal antibody B2D5 targeting BMP4 specifically includes using 200 U / mL collagenase IV to digest the IPSC clusters into relatively loose small cell clusters, placing them in a culture plate coated with Matrigel, and culturing them for 4 days in DMEM / F12 containing 100 ng / mL activin A, 1 M wortmannin, 10 μM Y-27632, and 100 μg / mL BMP4 monoclonal antibody B2D5 when 60% fusion is reached; and then continuing to place the cultured cells in a medium containing 2 μM RA, 20 ng / ml bFCF, and 50 ng / mL NOGGIN, 10μM Y-27632, 100μg / mL BMP4 monoclonal antibody B2D5 in F12 / IMDM and cultured for 4 days; then placed in 50ng / mL EGF, 10μM Y-27632, 100μg / mL BMP4 monoclonal antibody B2D5 in F12 / IMDM and cultured for 5 days to expand and differentiate pancreatic progenitor cells; the pancreatic progenitor cells were further induced for 11 days in DMEM high-glucose culture medium containing 1% ITS, 10 ng / ml HGF, 20mmol / L nicotinamide, 0.1mmol / L β-mercaptoethanol, 10μmol / L LY294002, and 30μmol / L B27 to obtain islet-like cells.

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