Cell composition and application thereof in preparation of preparation for treating diabetes

Abstract

The invention discloses a cell composition and application thereof in preparation of a preparation for treating diabetes mellitus. The cell composition contains pancreatic duct stem cells and functional islet cells formed by chemically inducing the pancreatic duct stem cells to differentiate. According to the invention, rat pancreatic duct stem cells derived from adult rats and functional islet cells chemically induced in vitro are transplanted through hepatic portal veins in a combined manner, so as to treat STZ modeling rat diabetes mellitus. Functional islet cells can directly and timely play a role in reducing blood sugar after entering the body; meanwhile, the transplanted pancreatic stem cells can be continuously amplified and gradually differentiated in vivo to form functional islet cells, so that the hypoglycemic effect is further continuously achieved, and diabetes can be treated for a longer time. The invention provides a method for treating diabetes mellitus through cell transplantation of human beings and other animals, and the method has important theoretical significance and huge social application value and economic value.

Description

Technical Field

[0001] The present invention relates to the field of diabetes, and more particularly, to a cell composition and application thereof in preparing a preparation for treating diabetes. Background Art

[0002] Diabetes is a major health hazard, yet traditional medications and insulin replacement therapy are ineffective in fundamentally curing the disease. Pancreatic stem cells, derived from adult stem cells in pancreatic tissue, possess self-renewal and multidirectional differentiation potential. Specifically, they can naturally differentiate into pancreatic cells, making them a promising candidate for diabetes treatment.

[0003] Studies have shown that isolating and culturing pancreatic stem cells in vitro can not only provide cell materials for studying pancreatic development, but also can induce them to differentiate into insulin-secreting cells for transplantation to treat diabetes. For example, using pancreatic cells isolated from prediabetic mice, pluripotent stem cells were induced to further differentiate into islet-like cell clusters that release insulin. Transplanting islet-like cell clusters can reduce blood sugar levels in non-obese diabetic mice and prevent diabetes (PMID: 9230348); epithelial-like stem cells derived from adult pancreas were cultured to form a monolayer, induced to form islet-like cell clusters, DTZ staining positive, and secreting insulin (PMID: 16003374); stem cells derived from human fetal pancreas were isolated and cultured, passaged for 50 generations, induced to differentiate into islet-like cell clusters, and transplanting islet-like cell clusters under the renal capsule had an anti-diabetic effect in rats (PMID: 1872 6523); in vitro induction and culture of human pancreatic stem cells led to differentiation into β-cell-like cells capable of secreting insulin (PMID: 20692413); pancreatic stem cells from fetal mice and pancreatic islets from adult rats were transplanted under the STZ-prepared kidney capsule, which had a short-term effect on the treatment of diabetes (Zhang Yusen, Bao Shi, Zhang Yue, et al., Transplantation of fetal mouse pancreatic stem cells and pancreatic islets to protect pancreatic islets. Chinese Journal of Tissue Engineering Research, 2014; 18(19): 3054-3059); stem cells from human pancreas were isolated and amplified and implanted into immunodeficient mice. These stem cells were able to differentiate into pancreatic tissue containing β-cells (PMID: 32354994).

[0004] At present, the technology of cell transplantation for the treatment of diabetes is still in the exploratory stage. Most studies are on inducing stem cells from different sources to differentiate into functional pancreatic islet cells in vitro for transplantation. These studies have the following problems: (1) The method of isolating and culturing pancreatic stem cells is not yet mature, and it is extremely difficult to obtain pancreatic stem cell lines from adults through isolation and culture; (2) The methods of inducing stem cells to differentiate into functional pancreatic β cells in vitro vary, and an efficient and stable chemical induction system has not yet been established; (3) The method of treating diabetes through cell transplantation is still in the exploratory stage, and the source, type, combination, ratio and number of transplanted cells, and transplantation site are different and have yet to be determined; (4) Most reports are on inducing stem cells to differentiate into functional pancreatic islet cells in vitro and then transplanting them. Although this overcomes the problem of insufficient pancreatic islet sources, the transplanted cells are already differentiated pancreatic islet cells, which have a limited lifespan and cannot function for a long time. Therefore, in clinical practice, a program for treating diabetes through cell transplantation has not yet been established. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a cell composition and its use in preparing a preparation for treating diabetes.

[0006] The first object of the present invention is to provide a cell composition.

[0007] The second object of the present invention is to provide the use of the composition in preparing a preparation for treating diabetes.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] The present invention utilizes isolated rat pancreatic ductal stem cells (deposited in the China Center for Type Culture Collection) and further in vitro chemical induction to obtain functional pancreatic islet cells. The rat pancreatic ductal stem cells and functional pancreatic islet cells are combined and transplanted via the rat portal vein into diabetic rats with loss of pancreatic islet structure and function induced by STZ. The results show that the transplanted cells can migrate to the rat pancreatic tissue, and the rat pancreatic ductal stem cells continuously proliferate and differentiate into β cells within the rat pancreatic tissue. Together with the transplanted in vitro induced functional pancreatic islet cells, they regenerate and reconstruct functional pancreatic islets to repair the pancreatic tissue damaged by STZ in the diabetic rats, causing pancreatic cells to express Pdx1 at the protein level and pancreatic islet cells to express insulin, and the expression of Pdx1 in the pancreatic tissue returns to normal levels. At the same time, the combined transplantation can also promote the recovery of serum insulin and C-peptide levels, blood HbA1c concentration, blood glucose concentration, body weight, food intake, and water intake of the diabetic rats to normal, indicating that pancreatic ductal stem cells and their induced pancreatic islet cells transplanted via the portal vein can cure STZ-induced diabetes in rats.

[0010] The results also showed that transplanting rat pancreatic ductal stem cells and functional islet cells in a specific ratio resulted in better results. In summary, the present invention provides a method for treating STZ-induced diabetes in rats by transplanting a pancreatic ductal stem cell line derived from adult rats and functional islet cells chemically induced in vitro using these cells via the hepatic portal vein. This invention provides a basis for cell transplantation therapy for diabetes in humans and other animals, and has important theoretical significance and enormous social application value.

[0011] The present invention therefore claims a composition comprising pancreatic ductal stem cells and functional pancreatic islet cells.

[0012] Preferably, the pancreatic ductal stem cells are isolated from pancreatic tissue.

[0013] More preferably, the pancreatic ductal stem cells are isolated from rat pancreatic tissue.

[0014] Further preferably, the pancreatic ductal stem cells are rat pancreatic ductal stem cells XMRPDSC2012, which were deposited in the China Center for Type Culture Collection on April 2, 2014, with a deposit number of CCTCC NO: C201457.

[0015] Preferably, the functional pancreatic islet cells are obtained by chemical induction using the pancreatic ductal stem cells.

[0016] More preferably, the induction medium used in the induction contains 10% FBS, 1% penicillin-streptomycin, 10mM NIC, 150nM INGAP, 1mM VitC, 4mM Ca 2+ The induction time was 28 days.

[0017] Preferably, the dosage ratio of the pancreatic ductal stem cells to the functional pancreatic islet cells is (0.9-1.1): (2.7-3.3), and the total dosage is (2.7-3.3)×10 6 cells.

[0018] More preferably, the ratio of the pancreatic ductal stem cells to the functional islet cells is 1:3, and the total amount is 3×10 6 cells.

[0019] Preferably, in a single dose of the cell composition, the amount of the pancreatic ductal stem cells and the functional islet cells are 0.25 mL and 3×10 6 cells / mL and 0.75mL 3×10 6 cells / mL.

[0020] Preferably, it contains a pancreatic ductal stem cell suspension and the functional pancreatic islet cell suspension.

[0021] The present invention also claims to protect the use of the composition in preparing a preparation for treating diabetes.

[0022] Preferably, the treatment of diabetes is restoration of one or more of the following: serum insulin and C-peptide levels, blood HbA1c concentration, blood glucose concentration, body weight, food intake and water intake to normal.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Using the isolated rat pancreatic ductal stem cells (China Center for Type Culture Collection, its collection number is CCTCC NO: C201457), a method for chemically inducing functional islet cells was established. On this basis, rat pancreatic ductal stem cells from adult rats and their in vitro chemically induced functional islet cells were transplanted together through the portal vein for the first time to treat STZ-modeled rat diabetes. Functional islet cells, after entering the body, can directly and promptly exert a hypoglycemic effect; at the same time, the transplanted pancreatic stem cells can continue to proliferate and gradually differentiate into functional islet cells in the body, further exerting a continuous hypoglycemic effect, and can cure diabetes for a longer period of time. The present invention provides a method for treating diabetes by transplanting cells from humans and other animals, which has important theoretical significance and huge social application value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 4: Growth of rat pancreatic ductal stem cells. A: Rat pancreatic ductal stem cells are polygonal and grow as monoclones. B: When the stem cells grow to 80% confluence, they appear like "paving stones."

[0026] Figure 2 The chromosome specimen and karyotype analysis of rat pancreatic ductal stem cells; A is the chromosome division phase; B is the chromosome karyotype diagram.

[0027] Figure 3 DTZ staining of pancreatic islets formed by induced differentiation of rat pancreatic ductal stem cells; A: stem cell-induced pancreatic islets stained positive for DTZ; B: primary rat pancreatic islets stained positive for DTZ.

[0028] Figure 4 The amounts of insulin and C-peptide released from pancreatic islet cells after glucose stimulation on day 28 of induction; Note: A is the amount of insulin released; B is the amount of C-peptide released; 5 mM represents low glucose; 25 mM represents high glucose; Significance analysis: *P < 0.05, **P < 0.01.

[0029] Figure 5 For induced pancreatic islet cell chromosome specimens and karyotype analysis; A is the chromosome division phase; B is the chromosome karyotype diagram.

[0030] Figure 6 This is a diagram of cell transplantation during rat portal vein surgery.

[0031] Figure 7 is the blood glucose concentration of rats in each group; A is a scatter plot of blood glucose concentration; B is a line graph of blood glucose concentration; 16.7 mmol / L is the blood glucose concentration criterion for judging diabetes in rats.

[0032] Figure 8 is the body weight of rats in each group; A is a scatter plot of body weight; B is a line graph of body weight.

[0033] Figure 9 is the food intake of rats in each group; A is a scatter plot of food intake; B is a line graph of food intake.

[0034] Figure 10 is the water intake of rats in each group; A is a scatter plot of water intake; B is a line graph of water intake.

[0035] Figure 11 The serum insulin and C-peptide release levels of rats in each group; A is the insulin release level at different time points; B is the C-peptide release level at different time points.

[0036] Figure 12 is the blood HbA1c concentration of rats in each group.

[0037] Figure 13 The effect of combined transplantation of group A cells on the pancreatic tissue morphology and structure of diabetic rats; scale bar = 200 μm, upper right corner is 200 times magnification; arrows point to pancreatic islets.

[0038] Figure 14 The figure shows the effect of combined transplantation of group A cells on the expression of insulin and Pdx1 proteins in the pancreatic tissue of diabetic rats; scale bar = 100 μm; red fluorescence indicates insulin expression, and green fluorescence indicates Pdx1 expression.

[0039] Figure 15 Western blot analysis of the effect of combined transplantation of group A cells on the expression of Pdx1 protein in pancreatic tissue of diabetic rats; Note: n=3, **P<0.01.

[0040] Figure 16 The purpose is to investigate the migration and colonization of pancreatic ductal stem cells and their induced islet cells in diabetic rats after transplantation via the portal vein. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0042] Example 1 Obtaining and karyotyping rat pancreatic ductal stem cells

[0043] 1. Experimental Methods

[0044] 1. Establishment of a pancreatic ductal stem cell line from adult rats

[0045] Referring to the method of prior art 1 (Isolation and Culture of Adult Rat Pancreatic Ductal Stem Cells [M], Dan Huiguo, 2013), a pancreatic ductal stem cell line derived from adult rats was established. The specific method is as follows:

[0046] Adult rat pancreatic tissue was used as the experimental material. Single pancreatic cells were obtained by digestion with 0.1g / mL type IV collagenase, and pancreatic ductal cells were isolated by Dextron dextran discontinuous density gradient centrifugation. Primary epithelial-like pancreatic ductal stem cells and a small number of fibroblasts adhered to the culture medium RPMI-1640 with 10% FBS, 0.11g / L sodium pyruvate, and 2.383g / L Hepes. Purified epithelial-like pancreatic ductal stem cells were obtained by digestion and passage with 0.25% trypsin and 0.04g / EDTA·2Na, gradually removing fibroblasts and other cells. Clusters of cells derived from single cells were selected by cloning rings and expanded and passaged in RPMI-1640 with 10% NBS and 10ng / mL EGF to establish an adult rat pancreatic ductal stem cell line.

[0047] The 23rd passage of this cell line was deposited in the China Center for Type Culture Collection on April 2, 2014, with a deposit number of CCTCC NO: C201457, and the culture name is rat pancreatic ductal stem cell XMRPDSC2012.

[0048] Adult rat pancreatic ductal stem cells were cryopreserved in liquid nitrogen using 80% RPMI-1640 + 10% FBS + 10% DMSO as the cell preservation medium. A programmed cooling cycle was used: 4°C (30 min) → -80°C, then 12 h → -196°C. The cells were rapidly thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 min, and the supernatant discarded. The cells were washed three times with PBS. A small amount of the cell suspension was collected and cell viability was calculated using a conventional cell counting method. Thawed survival was ≥90.8%.

[0049] Under an inverted microscope, pancreatic ductal stem cells adhered to the wall and showed a polygonal epithelial-like appearance. Their monoclonal growth characteristics were Figure 1 A in the figure; when grown to 80% confluence, it becomes a "paving stone" ( Figure 1 B) in.

[0050] After 1-2 days of subculture, the cells grew slowly and began to double in number after 3-4 days. The growth curve and subculture results showed that the adult rat pancreatic ductal stem cells had unlimited proliferation potential.

[0051] 2. Karyotype analysis

[0052] Refer to the conventional method, the specific steps are as follows:

[0053] Grow cells to 80% confluency, discard the culture medium, add 0.01% colchicine, and continue culturing for 4 hours. Digest with 0.25% trypsin, collect cells, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 0.5% KCl, mix well, and place in a 37°C water bath for hypotonicity for 5 minutes. Add 2 mL of methanol / glacial acetic acid (3:1) fixative, centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add the fixative again, and repeat the fixation twice. Discard the supernatant, leave 0.5 mL of chromosome suspension in the tube, and mix well. Take a portion of the chromosome suspension, drop it from a height of 50 cm onto a -20°C pre-cooled slide, and dry it. Stain with Giemsa for 10 minutes and dry it. Observe under a microscope, select well-dispersed metaphase chromosomes, take pictures, and perform karyotype analysis.

[0054] 2. Experimental Results

[0055] Chromosome specimens and karyotype analysis of rat pancreatic ductal stem cells Figure 2 The pancreatic ductal stem cells derived from adult rats are normal diploid cells with a chromosome number of 2n = 42. There are no chromosome breaks, deletions, translocations, or inversions, and no abnormalities in either number or structure.

[0056] Example 2 Chemically induced differentiation of pancreatic ductal stem cells into functional pancreatic islet cells

[0057] The rat pancreatic ductal stem cells prepared in Example 1 were 1×10 6 Cells were seeded into culture dishes at 100 cells / mL and expanded to a monolayer using RPMI-1640 cell culture medium containing 10% FBS, 10 ng / mL EGF, and 1% penicillin-streptomycin.

[0058] The monolayer of rat pancreatic ductal stem cells was replaced with an induction medium (containing 10% FBS, 1% penicillin-streptomycin, 10mM NIC (Nicotinamide), 150nM INGAP (Islet neogenesis associated protein), 1mM Vitamin C, 4mM Ca 2+ The cells were induced and cultured continuously for 28 days with the medium changed every other day to obtain functional pancreatic islet cells.

[0059] Monolayer rat pancreatic ductal stem cells were cultured in cell expansion medium (RPMI-1640 containing 10% FBS, 10 ng / mL EGF, and 1% penicillin-streptomycin) and the medium was changed every other day to serve as a negative control.

[0060] Comparative Example 1 Isolation of rat pancreatic islets

[0061] Take fresh pancreatic tissue from rats and cut into 1mm pieces. 3 , digested with 0.01% collagenase for 20 min, filtered through a 100-mesh filter, centrifuged at 1000 rpm for 5 min, and discarded the supernatant to obtain rat islets as a positive control. Example 3 DTZ Detection of Functional Islet Cells Formed by Chemically Induced Differentiation of Pancreatic Ductal Stem Cells

[0062] DTZ (dithizone, DTZ) is a cell viability dye that can react with Zn 2+ DTZ staining can be used as a marker for inducing stem cell differentiation into pancreatic β cells in vitro.

[0063] 1. Experimental Methods

[0064] The culture medium of the induced functional islet cells obtained in Example 2 was discarded, and the rat islets isolated from Comparative Example 1 were added with 1 mL of DTZ staining solution. The mixture was evenly mixed and transferred to a culture dish. The cells were stained at 37°C for 30 minutes. The DTZ staining solution was discarded, and the floating color was washed with PBS. The cells were observed under a microscope and photographed.

[0065] 2. Experimental Results

[0066] DTZ staining of rat pancreatic ductal stem cells induced by chemical differentiation to form functional pancreatic islet cells Figure 3 A in Example 2 was induced and cultured for 28 days, and differentiated into islet cell clusters, which contained a large amount of Zn 2+ β cells were stained reddish brown by DTZ.

[0067] Figure 3B in the figure shows directly isolated rat pancreatic islets, stained red-brown with DTZ.

[0068] The results showed that the functional pancreatic islet cells obtained in Example 2 included pancreatic β cells.

[0069] Example 4 Glucose stimulates pancreatic ductal stem cells to differentiate into functional islet cells that release insulin and C-peptide

[0070] 1. Experimental Methods

[0071] The culture medium of the functional pancreatic islet cells (induced group) and the monolayer of rat pancreatic ductal stem cells (negative control group) obtained in Example 2 was discarded and the cells were washed with PBS.

[0072] Three wells were randomly selected from each of the induction group and the negative control group for glucose stimulation. The cells were incubated at 37°C for 1 hour, and the supernatants were collected. Enzyme-linked immunosorbent assay (ELISA) was used to measure the release of insulin and C-peptide from the islet cell supernatants in response to different glucose concentrations (Insulin Kit Catalog No.: MM-0587R1; C-peptide Kit Catalog No.: MM-0588R1). The specific glucose stimulation method was as follows: for low glucose stimulation, 1 mL of 5 mM glucose was added to each well; for high glucose stimulation, 1 mL of 25 mM glucose was added to each well.

[0073] 2. Experimental Results

[0074] Different concentrations of glucose stimulate the pancreatic islets, and the release of insulin and C-peptide is shown in the figure. Figure 4 .

[0075] Compared with the negative control group, low glucose stimulation induced the release of insulin from pancreatic islet cells to be 3.11 times, and the release of C-peptide to be 2.42 times, with extremely significant differences (P < 0.01); high glucose stimulation induced the release of insulin from pancreatic islet cells to be 3.03 times, and the release of C-peptide to be 2.91 times, with extremely significant differences (P < 0.01).

[0076] Compared with low-glucose stimulation, high-glucose stimulation induced 1.21-fold higher insulin release and 1.31-fold higher C-peptide release in pancreatic islets, with both differences being extremely significant (P < 0.01). Furthermore, under glucose stimulation, insulin and C-peptide release were positively correlated.

[0077] This indicates that the functional islet cells obtained in Example 2 include functional β cells, which can secrete insulin and C-peptide, and the secretion capacity is positively correlated with the sugar concentration.

[0078] Example 5 Karyotype Analysis of Functional Islet Cells Formed by Chemically Induced Differentiation of Pancreatic Ductal Stem Cells

[0079] 1. Experimental Methods

[0080] The functional islet cells obtained in Example 2 were cultured, the culture medium was discarded, 0.01% colchicine was added, and the cells were cultured for another 4 hours. The cells were digested, collected, and chromosome specimens were prepared. The methods for preparing chromosome specimens and karyotype analysis were the same as those in Example 1.

[0081] 2. Experimental Results

[0082] The chromosome specimens and karyotype analysis of the functional pancreatic islet cells obtained in Example 2 are shown in Figure 5 It is a normal diploid cell with 42 chromosomes, 2n = 42, and no chromosome breakage, deletion, translocation, or inversion occurs, nor are there any abnormalities in chromosome number or structure.

[0083] Example 6 Preparation of rat diabetes model

[0084] Fifty normal adult male SD rats, weighing 250 ± 20 g, were purchased from the Guangdong Provincial Laboratory Animal Center. They were housed in a standard laboratory room at approximately 26°C and a relative humidity of 40–70%, with regular addition of chow, drinking water, and replacement of dry bedding.

[0085] Rats were housed conventionally for one week. Six rats were randomly selected as normal controls, and the remaining rats were intraperitoneally injected with a streptozocin (STZ) solution to establish a diabetic model. Rats were fasted for 14 hours before modeling, weighed, and intraperitoneally injected with 55 g / kg of 2% STZ solution. Seventy-two hours later, tail blood was drawn for blood glucose measurement for three consecutive days. Rats were considered diabetic if their blood glucose levels remained ≥16.7 mmol / L for three consecutive days and were accompanied by the "three mores and one less" symptom: elevated blood glucose, increased food and water intake, and decreased body weight.

[0086] Example 7 Pancreatic ductal stem cells and their induced functional islets transplanted into diabetic rats

[0087] 1. Experimental Methods

[0088] 1. Experimental groups and treatment methods

[0089] A total of 42 rats were tested, divided into 7 groups with 6 rats in each group (see Table 1 for details). The experimental period was 120 days.

[0090] Table 1:

[0091]

[0092] The specific methods of transplantation through the portal vein are:

[0093] Before the transplantation operation, the rats were weighed and recorded. The rats were anesthetized by intraperitoneal injection of 2.5% tribromoethanol at a dose of 300 mg / kg. The rats were restrained in the supine position, and the hair in the surgical area was shaved and disinfected. A 2 cm longitudinal incision was made in the skin along the midline from the xiphoid process downward, and the abdominal muscles and peritoneum were cut along the linea alba to expose the main trunk of the portal vein. The scalp needle was inserted into the portal vein about 5 mm, and the cell suspension or PBS was slowly injected at a dose of 1 mL / rat. The organs were repositioned, sutured, and the wound was disinfected. After the operation, 1 mL of 800,000 units of penicillin was injected subcutaneously. Pay attention to keeping warm. After the rats wake up, they are given adequate feed and diet. Figure 6 This is a diagram of the rat portal vein transplantation operation.

[0094] 2. Determination of blood glucose concentration, body weight, food intake and water intake of rats

[0095] Within 30 days after transplantation, fasting blood glucose concentration, body weight, food intake, and water intake of rats in each group were measured every 2 days at 8:00 am. 8 hours before measurement, rats in each group were deprived of food but provided with normal water.

[0096] Measure the above parameters every 10 days after 30 days of transplantation and every 20 days after 60 days of transplantation.

[0097] The specific method for detecting fasting blood glucose concentration is: puncture the tail vein of the test rat with a blood collection needle, and use a portable blood glucose meter to measure and record the blood glucose concentration.

[0098] The specific method for detecting food intake and water intake is: weigh the remaining mouse food and water bottle weight every day, subtract the weight of the mouse food and water bottle from the previous day, and record the food intake and water intake.

[0099] The mental state, fur color and bedding condition of the rats were observed, and the mortality rate of the rats was recorded.

[0100] 3. Detection of serum insulin and C-peptide release

[0101] On days 0, 30, 60, 90, and 120 after transplantation, three rats were randomly selected from each group and fasted for at least 8 hours. Blood was collected from the tail vein for fasting blood glucose measurement. Subsequently, 2 g / kg body weight of 20% glucose solution was administered orally. One hour after glucose administration, 0.5 mL of blood was collected from the tail vein and placed in a 1 mL centrifuge tube. The tubes were centrifuged at 3000 rpm for 10 minutes. Serum was then aspirated with a pipette, aliquoted, and stored at -20°C.

[0102] ELISA assays were performed according to the instructions of the rat insulin and C-peptide enzyme-linked immunosorbent assay kits to detect the secretion of insulin and C-peptide in rat serum. The rat insulin and C-peptide enzyme-linked immunosorbent assay kits were purchased from Jiangsu ELISA Biotechnology Co., Ltd.

[0103] 4. Glycated hemoglobin (HbA1c) testing

[0104] HbA1c is the product of the non-enzymatic reaction between red blood cell hemoglobin and serum sugars (primarily glucose). HbA1c levels are determined by historical, rather than immediate, blood glucose concentrations. It is generally believed that HbA1c levels effectively reflect average blood glucose levels over the past two to three months. HbA1c is often used clinically as a monitoring indicator for diabetes control. In this study, HbA1c levels in rats were measured 30 and 120 days after cell transplantation.

[0105] Specific measurement method: 30 days and 120 days after transplantation, 3 rats were randomly selected from each group each time, 0.5 mL of blood was collected from the tail vein, placed in a 1.5 mL centrifuge tube containing anticoagulant, and stored in a 4°C refrigerator.

[0106] The ELISA test was performed according to the instructions of the rat glycosylated hemoglobin (GHb) enzyme-linked immunosorbent assay kit to detect the glycosylated hemoglobin concentration in rat whole blood. The rat glycosylated hemoglobin (GHb) enzyme-linked immunosorbent assay kit was purchased from Jiangsu Enzyme Immunity Biotechnology Co., Ltd.

[0107] 4. Data processing and analysis

[0108] Data significance analysis was performed using IBM SPASS Statistics 26, inter-group comparisons were performed using one-way analysis of variance, and graphs were drawn using GraphPad Prism5.

[0109] 2. Experimental Results

[0110] 1. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on blood glucose in diabetic rats

[0111] Effects of pancreatic ductal stem cells and / or their induced functional islet cells on blood glucose in diabetic rats Figure 7 .

[0112] During the 120-day experiment, the blood glucose levels of the rats in the normal control group remained within the normal range of 5.08 ± 0.78 mmol / L. The blood glucose levels of the rats in the diabetic control group remained stable at 29.6 ± 2.84 mmol / L throughout their survival. However, due to the severity of the diabetes, the rats gradually died, and all of them had died by the 35th day.

[0113] Except for the stem cell transplantation group, the blood glucose concentrations of diabetic rats in other cell transplantation groups decreased rapidly from the first day of transplantation, and the difference was extremely significant compared with the diabetic control group (P < 0.01); at the 15th day after transplantation, there was no significant difference in the blood glucose concentration compared with normal rats (P > 0.05), and the ability of cell combined transplantation groups A and B to maintain normal blood glucose levels remained stable; the blood glucose concentration of rats in the stem cell transplantation group began to decrease on the 10th day after transplantation, reached the lowest on the 30th day, and then gradually increased; and the rats in this group died one after another due to long-term diabetes, and all died on the 60th day; the blood glucose concentration of rats in the combined transplantation group C began to decrease from the first day of transplantation, but it increased rapidly again after 20 days, and the rats died one after another, and all died on the 80th day.

[0114] The order of hypoglycemic effect of each cell transplantation group was combined transplantation group A, combined transplantation group B > induced islet cell transplantation group > combined transplantation group C > stem cell transplantation group.

[0115] This shows that combined transplantation group A and combined transplantation group B have a very significant improvement effect on hyperglycemia in diabetic rats caused by STZ, can restore the blood sugar concentration of diabetic rats to normal, and the blood sugar lowering function is sustained and stable.

[0116] The stem cell transplantation group and combined transplantation group C, which contained a higher proportion of pancreatic ductal stem cells and stem cells, showed the poorest glucose-lowering effects. This may be because stem cells require time to differentiate in vivo, preventing them from forming pancreatic β cells in time and releasing sufficient insulin to counteract the damage caused by STZ to the rats' pancreatic β cells.

[0117] 2. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on the body weight of diabetic rats

[0118] Effects of pancreatic ductal stem cells and / or their induced functional islet cells on body weight in diabetic rats Figure 8 .

[0119] During the 120-day experimental period, the body weight of rats in the normal control group steadily increased from 250g to 376.33±5.79g as they grew; the body weight of rats in the diabetic control group continued to decrease during their survival period, reaching 186.02±4.04g at 30 days.

[0120] During the first 8 days after transplantation, the body weights of diabetic rats in all cell transplantation groups decreased slowly, and there was no significant difference compared with the diabetic group. This was the effect of STZ-induced diabetes.

[0121] Eight days after transplantation, the body weight of rats in combined transplantation group A and combined transplantation group B continued to increase, and the difference was extremely significant compared with the diabetic group (P < 0.01); the body weight of rats in other transplantation groups first gradually increased, but began to decrease after 80 days in the induced islet transplantation group, 40 days in the combined transplantation group C, and 30 days in the stem cell transplantation group.

[0122] The order of effect of each cell transplantation group on the body weight of diabetic rats was combined transplantation group A and combined transplantation group B > induced islet transplantation group > combined transplantation group C > stem cell transplantation group. This indicates that combined transplantation groups A and B significantly improved the weight loss of diabetic rats induced by STZ, restoring their weight gain to normal levels and maintaining a stable state.

[0123] Throughout the experimental period, rats in the combined transplantation groups A and B consistently weighed less than those in the normal control group, likely due to the effects of preexisting diabetes and surgical transplantation. Rats in the induced islet transplantation group experienced weight loss after 80 days, likely due to the limited lifespan of the transplanted induced islet cells, which are already differentiated. The stem cell transplantation group and the combined stem cell transplantation group C showed the poorest weight gain, likely due to the high number or proportion of transplanted stem cells. This may be due to the time it takes for stem cells to differentiate in vivo, preventing them from forming pancreatic β cells in time and releasing sufficient insulin to counteract the damage caused by STZ to the rats' original pancreatic β cells.

[0124] 3. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on food intake in diabetic rats

[0125] Effects of pancreatic ductal stem cells and / or their induced functional islet cells on food intake in diabetic rats Figure 9 ,

[0126] During the 120-day experimental period, the food intake of rats in the normal control group increased steadily from 39.71±2.53g to 61.47±3.74g as they grew; the food intake of rats in the diabetic control group was about 60g for a long time during their survival.

[0127] On the day of transplantation (0d), the food intake of diabetic rats in all cell transplantation groups was very high, with no significant difference compared with the diabetic group (P>0.05), and extremely significant difference compared with the normal group (P<0.01), which was affected by STZ-induced diabetes.

[0128] Except for the stem cell transplantation group, the food intake of rats in all cell transplantation groups slowly decreased from the second day after transplantation. Compared with the diabetic control group, the differences ranged from significant to extremely significant (P < 0.05 to P < 0.01). 20 days after transplantation, the food intake gradually decreased to normal levels, with no significant difference compared with the normal group (P > 0.05), and remained consistent with the normal group. The food intake continued to increase as the rats grew.

[0129] The order of effect of each cell transplantation group on the food intake of diabetic rats was: combined transplantation group A, combined transplantation group B, induced islet transplantation group > combined transplantation group C > stem cell transplantation group. This indicates that combined transplantation groups A and B, as well as induced islet transplantation groups, significantly improved the STZ-induced increase in food intake in diabetic rats, restoring their food intake to normal levels.

[0130] 4. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on water intake in diabetic rats

[0131] Effects of pancreatic ductal stem cells and / or their induced functional islet cells on water intake in diabetic rats Figure 10 .

[0132] During the 120-day experimental period, the water intake of rats in the normal control group steadily increased from 54.92±7.27mL to 92.48±3.49mL as they grew, while the water intake of rats in the diabetic control group was always greater than 150mL during their survival.

[0133] On the day of transplantation (day 0), diabetic rats in all cell transplantation groups drank significantly more water, with no significant difference compared to the diabetic group (P>0.05) and a highly significant difference compared to the normal control group (P<0.01). This was attributed to the effects of STZ-induced diabetes. Subsequently, water intake in all cell transplantation groups, except the stem cell transplantation group, decreased rapidly. Twenty days after transplantation, water intake gradually returned to normal levels and remained stable, with no significant difference compared to the normal control group (P>0.05). The order of effect of each cell transplantation group on water intake in diabetic rats was: combined transplantation group A, combined transplantation group B, induced islet transplantation group > combined transplantation group C > stem cell transplantation group. This indicates that combined transplantation groups A, B, and induced islet transplantation groups significantly improved the increased water intake in diabetic rats induced by STZ, restoring water intake to normal levels.

[0134] 5. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on serum insulin and C-peptide release in diabetic rats

[0135] See the results Figure 11On the day of transplantation (0d), the levels of serum insulin and C-peptide released by diabetic rats in each cell transplantation group were not significantly different from those in the diabetic control group (P>0.05), but were extremely significantly lower than those in the normal control group (P<0.01). This further confirmed that intraperitoneal injection of STZ in rats greatly disrupted the function of pancreatic β cells in releasing insulin and C-peptide, and the rat diabetes model was successfully established.

[0136] Thirty days after transplantation, the release of serum insulin and C-peptide in rats of combined transplantation groups A and B and induced islet transplantation groups increased significantly compared with the diabetic group (P < 0.01), and there was no significant difference compared with the normal group (P > 0.05); while the release of serum insulin and C-peptide in other cell transplantation groups increased significantly (P < 0.05).

[0137] At 60, 90, and 120 days after transplantation, serum insulin and C-peptide release in rats in combined transplantation groups A and B was similar, with no significant differences compared to the normal control group (P>0.05). However, levels were significantly decreased in the induced islet transplantation group and combined transplantation group C (P<0.05), and extremely significantly decreased in the stem cell transplantation group (P<0.01). The order of cell transplantation-induced increases in serum insulin and C-peptide release in diabetic rats was combined transplantation group A and group B > induced islet transplantation group > combined transplantation group C > stem cell transplantation group. This suggests that combined transplantation groups A and B were the most effective, repairing STZ-induced damage to rat pancreatic β cells and restoring β-cell insulin and C-peptide release to normal levels. Furthermore, as stem cells continue to expand and differentiate into functional β cells, insulin and C-peptide release remains stable. The decreased release of insulin and C-peptide in the induced islet cell transplantation group may be due to the fact that the transplanted induced islet cells are differentiated cells, which gradually age over time and their ability to release insulin and C-peptide gradually decreases.

[0138] 6. Effects of pancreatic ductal stem cells and / or their induced functional islet cells on blood HbA1c concentration in diabetic rats

[0139] See the results Figure 12 On the 30th day after transplantation, the HbA1c concentration in the whole blood of rats in the normal control group was 3.4-4.1%. Compared with the normal control group, the HbA1c concentration in rats in the diabetic group was extremely significantly increased (P < 0.01). Compared with the diabetic group, the HbA1c concentration in rats in each cell transplantation group was extremely significantly decreased (P < 0.01).

[0140] On day 120 after transplantation, whole-blood HbA1c concentrations in rats in combined transplantation groups A and B, as well as in the induced islet transplantation group, decreased significantly compared to day 30. Furthermore, compared with the normal control group, HbA1c concentrations in combined transplantation groups A and B were closer, with no significant difference (P>0.05). This further demonstrates that STZ intraperitoneal injection of diabetic rats in this study stabilized the diabetic model, with rats in the diabetic control group maintaining elevated HbA1c concentrations over a long period. All cell transplantation groups were able to reduce HbA1c concentrations in diabetic rats to varying degrees, with combined transplantation groups A and B demonstrating the greatest hypoglycemic effect. Over time, elevated HbA1c concentrations in STZ-induced diabetic rats gradually decreased and returned to normal levels.

[0141] In summary, based on the measurement results of blood glucose concentration, body weight, food intake, water intake, serum insulin and C-peptide release, and blood HbA1c level of rats 120 days after transplantation, it was comprehensively judged that the anti-glycemic effect of cell combined transplantation group A and combined transplantation group B was better, and the best was combined transplantation group A.

[0142] Example 8 Histological morphology of diabetic rats transplanted with pancreatic ductal stem cells and their induced functional islet cells

[0143] 1. Experimental Methods

[0144] In Example 7, one rat was randomly selected from each of the normal control group, the diabetic group, and the combined transplantation group A at 30, 60, and 120 days after transplantation. The rats were sacrificed, and their pancreatic tissues were quickly removed, rinsed with PBS, and fixed with 4% formaldehyde for 24 hours. Paraffin sections of the rat pancreatic tissues were prepared as per standard procedures.

[0145] Prepare paraffin sections of pancreatic tissue: Dehydrate fixed pancreatic tissue in 80%, 95%, and 100% alcohol (30-45 min each cycle, followed by 30-45 min). Clear in xylene for 30-40 min, then embed in 62°C paraffin. Slice the tissue into 4 μm sections using a microtome, unfold in 47°C water, and mount on slides for later use.

[0146] Hematoxylin and Erythrocyte Staining: Dewax sections by exposing them to xylene and 50% ethanol-xylene solutions for 10 minutes each. Soak sections in 100%, 95%, 85%, and 70% alcohol for 2-5 minutes each, rehydrate, and rinse with distilled water for 2 minutes. Stain with hematoxylin for 10 minutes, rinse, separate sections with 1% hydrochloric acid-ethanol, and bluing with distilled water for 30 minutes. Stain with 0.5% eosin for 3 minutes, and dehydrate sections with 70%, 85%, 95%, and 100% alcohol for 2 minutes each. Clear sections with xylene twice for 5 minutes each and mount with neutral gum.

[0147] 2. Experimental Results

[0148] The results of H·E staining are shown in Figure 13 Thirty days after transplantation, the pancreatic islets in the normal control group were round or oval, with intact morphology and structure, and abundant and widely distributed. Compared with the normal control group, the diabetic rats had very few islets due to STZ damage, with a significant loss of islet cells. The islets became bundled or irregular in shape, with blurred boundaries with the surrounding tissue. Compared with the diabetic group, the diabetic rats in the combined transplant group A had numerous new islet-like cells in the pancreatic tissue, which were clustered into round or oval shapes. However, the number and size of the new islets were lower than those in the normal control group.

[0149] At 60 days after transplantation, the number of newly formed islets in the pancreatic tissue of rats in the combined transplantation group A increased, their size became larger, and their morphological structure became more complete.

[0150] At 120 days after transplantation, compared with the normal control group, the pancreatic tissue morphology and structure of the rats in the combined transplantation group A were basically restored, the islets were round or oval, the morphology and structure were complete, the islets were abundant and large in size.

[0151] The histological perspective further illustrates that after transplantation through the portal vein, pancreatic ductal stem cells and the functional islet cells induced by them can enter the pancreatic tissue and gradually repair the damage to the morphological structure of the pancreatic tissue islets caused by STZ through cell regeneration and islet reconstruction.

[0152] Example 9 Effect of combined transplantation of pancreatic ductal stem cells and their induced functional islet cells on the expression of insulin and Pdx1 proteins in pancreatic tissue of diabetic rats

[0153] 1. Experimental Methods

[0154] In each group of rats in Example 7, pancreatic tissue sections prepared at 30 days, 60 days, and 120 days after transplantation were subjected to fluorescent immunostaining for insulin and Pdx1. The immunofluorescence staining preparation method is as follows:

[0155] Prepared pancreatic tissue paraffin sections were placed in a 70°C slide oven for 2 hours and dewaxed in xylene three times for 10 minutes each. Sections were then immersed in 100%, 95%, 85%, 70%, and 50% alcohol solutions for 5 minutes each, rinsed sequentially, and microwave-unrecovered with EDTA antigen retrieval solution, cooled, and dried. Sections were permeabilized with 5% Triton X for 30 minutes, washed three times with PBS on a shaker for 5 minutes each, and blocked with 5% BSA at 37°C for 30 minutes. Sections were washed three times with PBS, then incubated with primary antibody dilution and incubated overnight at 4°C. Sections were washed three times with PBS on a shaker for 5 minutes each, then incubated with secondary antibody dilution at 37°C for 1 hour in the dark. Fluorescence was quenched with DAPI, nuclear staining was performed, and sections were mounted. Staining results were observed under an inverted fluorescence microscope and photographed. Rat pancreatic tissue was assayed for insulin and Pdx1 expression. Primary antibodies against insulin and Pdx-1, as well as secondary antibodies against rabbit and mouse goats, were purchased from Abcam.

[0156] 2. Experimental Results

[0157] Immunofluorescence staining results are shown in Figure 14 Thirty days after transplantation, pancreatic β cells in the pancreatic tissue of rats in the normal control group expressed insulin protein, showing red fluorescence; β cells and pancreatic progenitor cells widely expressed Pdx1, showing green fluorescence. Compared with the normal control group, diabetic rats showed no red fluorescence, indicating no β cells expressing insulin protein, nor green fluorescence, indicating no cells expressing Pdx1. Compared with the diabetic group, the pancreatic tissue of diabetic rats in the combined transplantation group A showed multiple areas of red fluorescence, indicating the presence of islets containing β cells. These β cells clustered in round or oval shapes, but the number and size of these islets were smaller than those in the normal control group. By day 60, the number of insulin-expressing β cells and the size of the islets in the pancreatic tissue of rats in the combined transplantation group A increased, and the number of cells expressing Pdx1 also increased. By day 120, the expression of insulin and Pdx1 in the pancreatic tissue of rats in the combined transplantation group A was essentially the same as that in the normal control group, with no significant difference. Insulin is a marker protein for β cells, while Pdx1 is expressed by both pancreatic progenitor cells and β cells. STZ-induced diabetic rats lack expression of these two proteins, indicating that STZ not only destroys pancreatic beta cells but also progenitor cells, completely impairing beta cell regeneration. Combined cell transplantation further demonstrated that after portal vein transplantation, pancreatic ductal stem cells and their induced islet cells can migrate to the pancreas. Under the influence of the pancreatic microenvironment, the stem cells can proliferate and differentiate into new functional islet cells. Together with the directly transplanted in vitro induced islet cells, they repair STZ-induced damage to pancreatic progenitor cells and islet cells, regenerate Pdx1 and insulin proteins, and exert their therapeutic effects on diabetes.

[0158] Example 10 Effect of combined transplantation of pancreatic ductal stem cells and their induced functional islet cells on the expression of Pdx1 protein in pancreatic tissue of diabetic rats

[0159] 1. Experimental Methods

[0160] Pancreatic tissues of rats in each group of rats in Example 7 were obtained from the diabetic control group (30 days of experiment), the normal control group (120 days of experiment), and the combined transplantation group A (120 days of experiment), and Western blot analysis was performed. The specific method is as follows:

[0161] Rat pancreatic tissue was placed in normal saline, rinsed several times, minced, and weighed. Lysis buffer containing phosphatase inhibitors was added according to tissue weight, and the mixture was homogenized for 2 minutes. Centrifuged at 1200 rpm for 5 minutes. The supernatant was collected and the total protein concentration was determined according to the BCA kit instructions. Loading buffer was added, the mixture was boiled at 100°C for 10 minutes, centrifuged for 30 seconds, and stored at -80°C until needed.

[0162] Slowly add the separation gel with a pipette, inject anhydrous ethanol to cover the gel surface, and let it stand at room temperature for 50 minutes. Pour off the anhydrous ethanol and absorb the remaining liquid with filter paper, insert the comb, and let it stand at room temperature for 30 minutes. After the stacking gel is completely solidified, fix it in the electrophoresis tank, and add electrophoresis buffer to the inner and outer tanks. Pull out the comb, mix the protein sample with 5× SDS buffer, add it to the well, and add 5μL protein marker to the first well. Place the electrophoresis tank on ice to maintain a low temperature environment, set the stacking gel stage to 80V electrophoresis for 20 minutes, and adjust it to 120V after the sample enters the separation gel and continue electrophoresis until the marker bands are fully separated.

[0163] After electrophoresis, remove the gel, cut out the band containing the target protein, and soak it in transfer buffer for 5 minutes. At the same time, cut the PVDF membrane and activate it with methanol for 3 to 5 minutes to make it translucent. Place the soaked sponge, three layers of filter paper, gel, PVDF membrane and filter paper sponge in the transfer folder in sequence, ensuring that there are no bubbles between the layers to form a "sandwich" structure. Place it in the transfer tank, add transfer buffer, ice bath, set a constant current of 300mA to transfer for 1 hour. Remove the PVDF membrane and place it in blocking solution. Block overnight at 4°C or block on a shaker at room temperature for 4 hours. Discard the blocking solution and wash 3 times with TBST, each for 10 minutes.

[0164] Immerse the membrane in Pdx1 antibody dilution buffer and incubate at 37°C on a shaker for 2 hours. Wash the membrane three times with TBST (10 minutes each). Place the membrane in goat anti-rabbit secondary antibody dilution buffer and incubate at 37°C on a shaker for 2 hours. Wash the membrane three times with TBST (10 minutes each). In the dark, mix ECL luminescent solution A and solution B in equal proportions and evenly drip onto the PVDF membrane. Let it sit for 2 minutes to allow for full signal reaction. Remove excess luminescent solution with filter paper and perform imaging using an automated chemiluminescence imaging system. Images were analyzed using Image J software to analyze the grayscale values ​​of the bands and assess the expression level of the target protein.

[0165] 2. Experimental Results

[0166] Western blot test results are shown in Figure 15 At 120 days after transplantation, pancreatic cells in the normal control group expressed a certain amount of Pdx1 protein. Compared with the normal control group, Pdx1 expression in the pancreatic cells of the diabetic control group was significantly decreased (P < 0.05). Compared with the diabetic control group, Pdx1 expression in the combined transplantation group A was significantly increased (P < 0.05); there was no significant difference compared with the normal control group (P > 0.05). Pdx1 is a key regulator of pancreatic β-cell development and function. Results in the diabetic control group confirmed that STZ inhibits the development and function of pancreatic cells, particularly pancreatic islet cells (including β-cells), by reducing the key regulator Pdx1. Results in the combined cell transplantation group A further confirmed that transplantation via the portal vein resulted in cell migration to the pancreatic tissue, stem cell proliferation, and differentiation into β-cells. Combined with the transplantation of induced pancreatic islet cells containing β-cells, Pdx1 protein production in diabetic rats was upregulated, and expression was even restored to normal levels, effectively curing STZ-induced diabetes in rats.

[0167] Example 11 Migration and colonization of pancreatic ductal stem cells and their induced functional islet cells in diabetic rats

[0168] PKH26 stably binds to lipids in the cell membrane and is transferred to daughter cells during cell division, allowing the labeled cells to maintain their fluorescent signal throughout their growth cycle. The dye has an excitation wavelength of approximately 551 nm and an emission wavelength of approximately 567 nm. Labeled cells exhibit an orange-red fluorescence under a fluorescence microscope.

[0169] 1. Experimental Methods

[0170] Cell labeling: The rat pancreatic ductal stem cell suspension prepared in Example 1 and the functional pancreatic islet cell suspension prepared in Example 2 were mixed at a volume ratio of 1:3, wherein: 0.25 mL of the rat pancreatic ductal stem cell suspension prepared in Example 1 and 3×10 6cells / mL, 0.75 mL of the functional islet cell suspension obtained in Example 2 3×10 6 10 cells / mL. Centrifuge, discard the supernatant, wash twice with PBS, add 1 mL of PKH26 staining solution, and incubate at 37°C for 15 min. Centrifuge, discard the staining solution, and wash twice with PBS, each for 5 min. Observe the labeling under a microscope, collect the cells, and protect from light. The labeled cells are ready for use.

[0171] Transplanted rats: The labeled cells were transplanted into the diabetic rats of Example 6 via the portal vein according to the method of Example 7.

[0172] Sampling: Rats were killed 12 h, 24 h, 3 d, 5 d, and 7 d after transplantation, and liver, kidney, heart, pancreatic head, pancreatic body, and pancreatic tail samples were quickly taken, quickly rinsed with PBS, snap-frozen in liquid nitrogen, placed in labeled EP tubes, and stored at -80°C.

[0173] Prepare frozen sections: Place the tissue block in a section embedding cassette, add OTC compound to cover the tissue, and place on the freezing table of a cryostat for rapid freezing. Once the OTC compound turns white and hardens, slice the tissue to a thickness of 4-5 μm, mount the slide, and seal the slide. Observe under a fluorescence microscope and photograph the tissue.

[0174] 2. Experimental Results

[0175] See the results Figure 16 At 12 hours, 24 hours, and 3 days after transplantation, a small number of PKH26-labeled pancreatic ductal stem cells and their induced islet cells appeared in each organ. At 5 and 7 days after transplantation, the labeled cells migrated and homed primarily to the pancreatic tissue via the blood circulation. On the 7th day, the pancreatic tail (F5) had the largest number of homing cells. Therefore, after transplantation via the portal vein, pancreatic ductal stem cells and their induced islet cells can migrate to the heart, liver, kidney, and pancreas through the blood circulation, but the vast majority of transplanted cells homed to the pancreatic tissue, primarily colonizing the pancreatic tail. The migration and colonization of pancreatic ductal stem cells and their induced islet cells may be due to the secretion of chemokines by damaged pancreatic cells in diabetic rats, which promotes their homing.

Claims

1. A cell composition, characterized in that Contains pancreatic ductal stem cells and functional pancreatic islet cells.

2. The composition according to claim 1, characterized in that The pancreatic ductal stem cells are isolated from pancreatic tissue.

3. The composition according to claim 2, characterized in that The pancreatic ductal stem cells are isolated from rat pancreatic tissue.

4. The composition according to any one of claims 1 to 3, characterized in that The pancreatic ductal stem cells are rat pancreatic ductal stem cells XMRPDSC2012, which were deposited in the China Center for Type Culture Collection on April 2, 2014, with a deposit number of CCTCC NO: C201457.

5. The composition according to any one of claims 1 to 4, characterized in that The functional pancreatic islet cells are obtained by chemical induction using the pancreatic duct stem cells.

6. The composition according to claim 5, characterized in that The induction medium used in the induction was 10% FBS, 1% penicillin-streptomycin, 10 mM NIC, 150 nM INGAP, 1 mM VitC, 4 mM Ca 2+ The induction time was 28 days.

7. The composition according to claim 1, characterized in that The dosage of the pancreatic ductal stem cells and the functional pancreatic islet cells is (0.9-1.1): (2.7-3.3), and the total dosage is (2.7-3.3)×10 6 cells.

8. The composition according to claim 1, characterized in that Contains pancreatic duct stem cell suspension and functional pancreatic islet cell suspension.

9. Use of the composition according to claim 1 in preparing a preparation for treating diabetes.

10. The use according to claim 9, characterized in that The treatment of diabetes is to restore one or more of the following to normal: serum insulin and C-peptide levels, blood HbA1c concentration, blood glucose concentration, body weight, food intake and water intake.

Images