WO2022143905A1 - 一种治疗糖尿病的药物及其方法 - Google Patents

一种治疗糖尿病的药物及其方法 Download PDF

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WO2022143905A1
WO2022143905A1 PCT/CN2021/143115 CN2021143115W WO2022143905A1 WO 2022143905 A1 WO2022143905 A1 WO 2022143905A1 CN 2021143115 W CN2021143115 W CN 2021143115W WO 2022143905 A1 WO2022143905 A1 WO 2022143905A1
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stem cells
mesenchymal stem
implantation
pancreatic
diabetes
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PCT/CN2021/143115
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English (en)
French (fr)
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寇晓星
施松涛
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医微细胞生物技术(广州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to a medicine for treating diabetes and a method thereof.
  • Diabetes is a disease that develops from insufficient insulin and its loss of function. Once it develops, it is difficult to cure. Diabetes can be mainly divided into two types: type I diabetes (insulin-dependent diabetes) and type II diabetes (non-insulin-dependent diabetes).
  • Type II diabetes is a chronic disease that develops from insulin resistance, it becomes a problem related to lifestyle habits such as obesity due to overeating and inactivity, stress, etc.
  • Type II diabetes usually develops in middle-aged and elderly people, and many People with diabetes have type II diabetes.
  • Type I diabetes is a chronic disease caused by autoimmune diseases, viral infections, etc. that destroy pancreatic cells (also called insulin-producing cells) to stop insulin secretion in the body.
  • diabetes In recent years, the incidence of diabetes is on the rise, and it tends to be younger. Diabetes has become a common endocrine and metabolic disease that seriously endangers human health. Different types of diabetes cause the beta cells in the pancreas to fail to produce enough insulin to lower blood sugar levels, leading to hyperglycemia.
  • the current treatment methods are mainly drug therapy. Although drug therapy can lower blood sugar, it cannot cure diabetes. Insulin therapy can control symptoms, but long-term use can lead to insulin resistance. Pancreatic islet transplantation is an effective means of curing diabetes with low risk. Diabetes is a major public health problem due to its widespread prevalence and lack of a cure.
  • the present disclosure provides the use of mesenchymal stem cells or pancreatic exocrine gland tissue derived from exocrine pancreas in the preparation of a drug for treating/preventing diabetes.
  • the pancreatic exocrine gland tissue is generated from mesenchymal stem cells of the pancreatic exocrine gland.
  • the mesenchymal stem cell drug of exocrine pancreas is an injectable preparation.
  • the mesenchymal stem cell drug of exocrine pancreas is a subcapsular injection preparation.
  • the present disclosure provides the use of substances promoting the formation of pancreatic exocrine glands, which are naturally differentiated through ectopic regeneration of pancreatic exocrine gland-derived mesenchymal stem cells, in the preparation of a drug for treating/preventing diabetes.
  • pancreas has endocrine glands (endocrine cells) and exocrine glands (exocrine cells), and is an organ that plays an important role in these two secretory cells.
  • Exocrine cells mainly function to secrete digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase.
  • the diabetes is Type I diabetes.
  • the present disclosure provides a system for treating/preventing diabetes, comprising mesenchymal stem cells of the exocrine pancreas, and an implantable device.
  • the implantation device is used to implant the mesenchymal stem cells of the exocrine pancreas into a specific site in a patient.
  • the interventional device is a trocar.
  • the targeted site includes subrenal capsule, subcutaneous, or mesentery.
  • the system further comprises an ultrasound device or an endoscopic device for guiding the positioning of the trocar.
  • the implant is a subcapsular implant.
  • the site of ectopic regeneration includes subcapsular, subcutaneous.
  • pancreatic islets are necrotic and do not produce insulin, and there is currently no cure.
  • MSCs can promote islet cell regeneration, and MSC transplantation is expected to become a new method for the treatment of diabetes, especially for the treatment of type 1 diabetes.
  • mesenchymal stem cells can treat diabetes by regenerating pancreatic ⁇ cells.
  • pancreatic stem cells can differentiate into insulin-secreting islet-like structures, and the sequential transplantation of pancreatic islets and pancreatic stem cell-derived islet-like structures has therapeutic effects on diabetes in rats. They also found that MSCs with multi-directional differentiation potential can also differentiate into insulin-producing cells (IPCs) and vascular endothelial cells in vivo. Generation, and then the proliferation of residual islets, this study was confirmed by Barky et al. In addition, the study of Arzouni et al.
  • MSC transplantation can improve human islet function by releasing annexin A1 (ANXA1) and extracellular matrix.
  • ANXA1 annexin A1
  • co-transplantation of autologous MSCs and islets can improve the success rate and safety of islet transplantation, effectively reduce allograft immune rejection and early graft loss caused by inflammatory responses, or provide treatment for type 1 diabetes. new program.
  • Another example is the study by Daisong Wang et al. that islet progenitor cells can form islet-like tissue, in which islet ⁇ cells dominate the tissue. Long-term culture of this tissue can reverse diabetes.
  • pancreatic mesenchymal stem cells into animals can achieve the effect of treating diabetes by regenerating pancreatic exocrine glands instead of islet cells, and its treatment is very significant.
  • the inventors have found that PMSCs express high levels of IL-6 by inhibiting IL-17 to improve the immune microenvironment of the pancreas, thereby rescuing damaged islet cells. Furthermore, we use a knockout mouse model to show that lack of IL-6 leads to increased severity of STZ-induced type 1 diabetes and is resistant to treatment with PMSC engraftment, thus confirming that PMSCs act on IL-6 to protect ⁇ cell.
  • the present disclosure also provides a method of obtaining tissue of the exocrine pancreas formed by implantation of mesenchymal stem cells.
  • the tissue of the extrapancreatic gland is formed by ectopic implantation of mesenchymal stem cells into the animal.
  • the animal is selected from the group consisting of pig, rat, mouse, hamster, rabbit, pig, cow, deer, sheep, goat, chicken, cat, horse, dog, orangutan, monkey. In some embodiments, the animal is selected from mice.
  • the implantation includes subrenal capsule implantation, subcutaneous implantation, mesenteric implantation, and the like.
  • the implantation is a subcapsular implantation.
  • the tissue of the extrapancreatic glands is implanted in a non-living tissue model.
  • the mesenchymal stem cells include pancreatic mesenchymal stem cells, bone marrow mesenchymal stem cells.
  • the mesenchymal stem cells are pancreatic mesenchymal stem cells.
  • the inventors have found that subrenal engraftment of PMSCs shows a significant sustained therapeutic effect in STZ-induced mice compared to intravenous infusion of PMSCs.
  • the present disclosure reveals the unknown role of exocrine pancreatic regeneration in protecting beta cells and demonstrates a soil rescue seed strategy for type 1 diabetes treatment.
  • the mesenchymal stem cells are free mesenchymal stem cells.
  • the mesenchymal stem cells are directly implanted without treatment.
  • mesenchymal stem cells are used to treat diabetes, and the mesenchymal stem cells are generally processed and activated in vitro.
  • BEHROUS DAVANI et al. studied human islet-derived mesenchymal stem cells for the treatment of type I diabetes after obtaining cells from islets, they were expanded in vitro using growth medium, and differentiation induction was added to the expanded mesenchymal stem cells.
  • BSA bovine serum albumin
  • Fraction V Fatty Acid Free MP Biomedicals, Irvine, CA, http:/ /www.mpbio.com
  • Gabco 1 ⁇ insulin-transferrinselenium-A
  • Human Islet-Derived Precursor Cells are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone-Expressing Cells In Vivo. Stem Cells, 2007, 25:3215–3222.).
  • Another example is Daisong Wang et al. by flow sorting Procr + pancreatic stem cells and endothelial cells co-cultured in vitro, expanded for a period of time and then implanted in vivo to form islet tissue, transplanted into mice, and reversed diabetes (Daisong Wang, et al. Long -Term Expansion of Pancreatic Islet Organoids from Resident Procr+Progenitors. Cell, 2020, 180:1198–1211.).
  • the mesenchymal stem cells conventionally cultured in vitro can be directly digested into single free cells, and then directly implanted into animals to achieve therapeutic effects, which has very good operability and is easy to popularize and use.
  • the mesenchymal stem cells can be mixed with scaffold materials such as gelatin sponge.
  • the culture-expanded pancreatic-derived mesenchymal stem cells express various pancreatic progenitor markers PDX1, Nkx6.1, Ptf1a, Hnf1b and Ngn3.
  • the present disclosure provides a method for treating diabetes, the method for treating diabetes comprising the steps of: (1) culturing mesenchymal stem cells with a culture medium; (2) removing the mesenchymal stem cells in step (1). (3) After suspending the mesenchymal stem cells collected in step (2) with PBS or physiological saline (but not limited to this), implant the animal kidney capsule Subcutaneous, subcutaneous or mesenteric, so as to achieve therapeutic effect.
  • the method for obtaining pancreatic exocrine gland tissue comprises the following steps: (1) culturing the mesenchymal stem cells with a culture medium; (2) removing the culture medium of the mesenchymal stem cells in step (1), Collect mesenchymal cells; (3) after suspending the mesenchymal stem cells collected in step (2) with PBS or physiological saline (but not limited to this), implant them under the renal capsule, subcutaneously or on the mesentery of the animal , thereby obtaining pancreatic exocrine tissue.
  • the PBS is a trace amount of PBS.
  • the mesenchymal stem cells can be mixed with scaffold materials such as gelatin sponge.
  • the components of the culture medium in step (1) include FBS, glutamine (200 mM), 2-ME and ⁇ -MEM.
  • the components of the culture medium in step (1) include FBS, penicillin/streptomycin solution, glutamine (200 mM), 2-ME (55 mM), ⁇ -MEM.
  • the mesenchymal stem cells are cultured to generation P1-P3, but not limited thereto.
  • the present disclosure provides a pancreatic exocrine gland tissue obtained by the method.
  • the present disclosure provides the tissue of the exocrine pancreas in the discovery and screening of therapeutic drugs for pancreatic-related diseases, or the toxicity assay of therapeutic drugs for pancreatic-related diseases, or pancreatic embryology, or pancreatic cell lineage and differentiation Pathway studies, or gene expression studies, or mechanisms involved in pancreatic damage and repair, or inflammatory diseases of the pancreas, or in vitro use in the study of therapeutic mechanisms.
  • pancreatic exocrine gland tissue in recombinant gene expression.
  • the tissue of the exocrine pancreas is used in the preparation of a medicament for the treatment of a pancreatic disorder or disease, or in the preparation of a medicament for regenerative medicine.
  • the pancreatic disease comprises diabetes.
  • the diabetes is preferably type I diabetes.
  • composition comprising IL-6 and mesenchymal stem cells in the manufacture of a medicament for treating and/or preventing diabetes.
  • the source of the mesenchymal stem cells includes bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, peripheral blood, or pancreas.
  • the source of the mesenchymal stem cells is bone marrow.
  • the bone marrow mesenchymal stem cells are free bone marrow mesenchymal stem cells.
  • the IL-6 and mesenchymal stem cells are administered simultaneously.
  • the IL-6 and mesenchymal stem cells are administered alone.
  • IL-6 is administered first, followed by mesenchymal stem cells.
  • the mesenchymal stem cells are administered first, followed by the administration of IL-6.
  • the medicament is an injectable formulation.
  • the drug is selected from intravenous, intramuscular, subcutaneous, or intrathecal injections.
  • the mesenchymal stem cells are subcapsular injections.
  • the IL-6 is an intraperitoneal injection.
  • the medicament further includes a pharmaceutically acceptable pharmaceutical carrier.
  • the pharmaceutically acceptable carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.
  • the diabetes is Type I diabetes.
  • the inventor's research found that the effect of bone marrow mesenchymal stem cells in the treatment of type 1 diabetes is far less than that of pancreatic exocrine gland-derived mesenchymal stem cells, and that bone marrow mesenchymal stem cells are co-cultured (direct contact) with pancreatic islet ⁇ cells, and bone marrow mesenchymal stem cells are in direct contact with each other. Stem cells have a killing effect on islet ⁇ cells. This leads to the fact that bone marrow mesenchymal stem cells are not an ideal cell source for the treatment of type 1 diabetes.
  • the inventors are not limited to this status quo, but through continuous exploration and research, they found that after adding IL-6 to bone marrow mesenchymal stem cells, it was unexpectedly found that the effect of treating type 1 diabetes can be greatly improved. Thereby, the technical obstacle of the poor effect of the bone marrow mesenchymal stem cells in the treatment of type 1 diabetes is overcome, and the source of cells for the treatment of type 1 diabetes is expanded. Compared with pancreatic exocrine gland-derived mesenchymal stem cells, bone marrow mesenchymal stem cells are more easily obtained, and a wide range of cells can be used in clinic. It has very good practical significance.
  • the present disclosure provides a system for treating/preventing diabetes, comprising mesenchymal stem cells and IL-6, and an implantable device.
  • the implantation device is used to implant mesenchymal stem cells at a specific site in a patient.
  • the implant device is a trocar.
  • the site of targeting comprises subrenal capsule, subcutaneous, or mesentery.
  • the system further comprises an ultrasound device or an endoscopic device.
  • the ultrasound device or endoscopic device is used to guide the positioning of the implant device.
  • the present disclosure provides a method of treating diabetes comprising administering to a patient mesenchymal stem cells derived from pancreatic exocrine glands, or transplanting said pancreatic exocrine gland tissue, or administering to a patient IL-6 and mesenchymal stem cells composition of stem cells.
  • the diabetes is Type I diabetes.
  • the mesenchymal stem cells of the exocrine pancreas are administered to the patient by subrenal capsule implantation, subcutaneous implantation, or mesenteric implantation.
  • the implantation is an ectopic implantation.
  • the ectopic implant is a non-living tissue model implant.
  • the source of the mesenchymal stem cells comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, peripheral blood or pancreas.
  • the bone marrow mesenchymal stem cells are free bone marrow mesenchymal stem cells.
  • the IL-6 and mesenchymal stem cells are administered simultaneously.
  • the IL-6 and mesenchymal stem cells are administered alone.
  • IL-6 is administered first, followed by mesenchymal stem cells.
  • the mesenchymal stem cells are administered first, followed by the administration of IL-6.
  • the composition is an injectable formulation.
  • the composition is selected from intravenous injection, intramuscular injection, subcutaneous injection, or intrathecal injection.
  • the mesenchymal stem cells are subcapsular injections.
  • the IL-6 is an intraperitoneal injection.
  • the composition further includes a pharmaceutically acceptable pharmaceutical carrier.
  • the pharmaceutically acceptable carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.
  • 1A-1G are identification of murine PMSCs.
  • (1A) Flow cytometry analysis confirmed that CD105 + PMSCs co-express the ⁇ -cell marker insulin, acinar cell marker amylase, ductal cell marker CK19 and pancreatic progenitor cell markers Ngn 3, Ptf1a, Hnf1 ⁇ , PDX1 and Nkx6. 1.
  • (1B) Flow cytometry analysis showed that PMSCs expressed mesenchymal stem cell surface markers Sca-1, CD105, CD73, CD44, while blood cell markers CD34 and CD45 were negative.
  • Figures 2A-2F illustrate the regeneration of ectopic exocrine pancreas from pancreatic stem cells.
  • Immunofluorescence showed double staining for insulin and amylase in the ectopic regenerating pancreas.
  • the right panel is a higher magnification view of the immunostained image from the boxed area in the left H&E image.
  • the black dashed line indicates the junction of the kidney and the regenerating exocrine pancreas.
  • the yellow dotted line indicates the exocrine pancreatic acini.
  • Kidney Kidney, EP: Heterotopic Pancreas. (2E) H&E and immunofluorescence staining of normal and ectopic regenerating pancreas from wild-type mice.
  • H&E staining of ectopic regenerated pancreas shows typical staining of exocrine pancreatic compartments, similar to normal pancreas.
  • Immunofluorescence assays showed negative staining for endocrine cell markers in the ectopic regenerated pancreas, such as insulin for ⁇ cells, glucagon for ⁇ cells, somatostatin for ⁇ cells, and pancreatic polypeptide staining for PP cells; Alveolar and ductal cell markers such as amylase, trypsin, pancreatic lipase, and CK19 stained positive.
  • the right panel is a higher magnification view of the boxed area in the left H&E and immunostained images.
  • the white dashed line indicates the junction of the kidney with the regenerating exocrine pancreas.
  • the yellow dotted line indicates the exocrine pancreatic acini.
  • Black dashed lines indicate catheters.
  • EP Ectopic Pancreas. Scale bar, low magnification is 200 ⁇ m and high magnification is 50 ⁇ m.
  • FIGS. 3A-3M show that PMSCs-mediated subcapsular pancreatic regeneration ameliorates STZ-induced type I diabetes.
  • STZ low-dose streptozotocin
  • IPGTT intraperitoneal glucose tolerance test
  • 3E Representative H&E and immunofluorescence images showing that subrenal capsule implantation of PMSCs regenerates an ectopic exocrine pancreas in type 1 diabetic mice in which no insulin-positive cells can be detected.
  • 3F Implantation of PMSCs significantly improved the survival of type 1 diabetes induced by a single high-dose injection of STZ (200 mg/kg body weight) in immunodeficient mice.
  • KC Subcapsular implantation of MSCs.
  • 3J-3L Renal subcapsular implantation of PMSCs resulted in greater improvements in glucose clearance, serum C-peptide and insulin levels compared with systemic infusion of PMSCs.
  • Figures 4A-4F show that subrenal capsule implantation of PMSCs rescues damaged orthotopic pancreas/islets in type 1 diabetic mice.
  • PMSC-KC sub-renal capsule implantation of PMSCs
  • Figures 5A-5B are co-staining of the pancreatic exocrine gland specific markers trypsin and pancreatic lipase with the beta cell marker insulin.
  • Subrenal engraftment of PMSCs significantly rescued STZ-induced damaged insulin-positive ⁇ cells and trypsin- and pancreatic lipase-positive acinar cells compared with BMMSCs implantation or systemic infusion of PMSCs. Scale bar, 50 ⁇ m. All results represent data generated in at least three independent experiments.
  • FIGS. 6A-6D show that PMSCs express high levels of IL-6 to protect islet beta cells.
  • 6A The level of IL-6 secreted by PMSCs was significantly higher than that of BMMSCs.
  • Cytokine array analysis of culture supernatants from mouse PMSCs and BMMSCs. Equal volumes of PMSC and BMMSC culture supernatants were taken for cytokine arrays (n 3).
  • 6B-6D ELISA, PCR and Western blot analysis confirmed that PMSCs expressed higher levels of IL-6 than BMMSCs.
  • FIGS 8A-8D are PMSCs expressing high levels of IL-6 to protect beta cells.
  • 8A Flow cytometry analysis showed that most CD105 + PMSCs expressed IL-6.
  • 8B Immunostaining of pancreatic tissue showed that IL-6 was detected in both exocrine and endocrine tissues.
  • FIGS. 9A-9E explore the pivotal role of IL-6 in PMSC-mediated therapy in STZ-induced diabetic mice.
  • IPGTT intraperitoneal glucose tolerance test
  • 9C The IL-6 knockout PMSCs implanted under the renal capsule locally regenerated acinar structures, and basophilic stained acinar-like cells could be detected scattered within the cells.
  • (9D, 9E) Histological and immunostaining analyses further confirmed that IL-6 knockout PMSC implantation did not rescue the number of damaged islets, insulin-positive cells in pancreatic tissue of STZ-induced diabetic mice
  • Figure 10 shows that PMSCs express high levels of IL-6 to protect beta cells.
  • the ability of IL-6-knockout PMSCs to protect MIN6 ⁇ cells from STZ-induced apoptosis was reduced in vitro.
  • MIN6 ⁇ cells were co-cultured with wild-type or IL-6 knockout PMSCs after 5-10 mg/ml STZ treatment for 24 hours.
  • Apoptosis of MIN6 cells was measured by labeling with annexin V and 7AAD using flow cytometry.
  • FIGS 11A-11D show that subcapsular implantation of PMSCs increases IL-6 levels.
  • 11A Subrenal engraftment of PMSCs expresses high levels of IL-6. Representative immunofluorescence staining showed strong IL-6 immunostaining in the wild-type PMSCs-implanted group. No IL-6 staining was observed in the IL-6 knockout PMSCs implanted group.
  • 11B ELISA analysis showed that serum IL-6 levels increased at 2 and 4 weeks after PMSCs implantation. The dotted line represents the basal serum IL-6 level of the control mice.
  • 11C Implantation of IL-6 knockout PMSCs failed to suppress IL-17 levels.
  • IL-10, IL-17, TGF- ⁇ and IL-6 in orthotopic pancreas were analyzed by ELISA.
  • 11D Implantation of IL-6 knockout PMSCs failed to inhibit Th17 cell activation in a system of type 1 diabetic mice.
  • CD4 + IL-17 + Th17 cells and CD4 + T cells in the spleen of type 1 diabetic mice were shown to be CD25 + Foxp3 + Treg cells by flow cytometry analysis. All results represent data generated in at least three independent experiments.
  • Figures 12A-12D show that IL-6 knockout mice exhibit resistance to PMSCs implantation therapy.
  • (12C, 12D) Representative H&E images of host orthotopic pancreas/islets after subrenal capsule implantation of PMSCs in type 1 diabetic IL-6 knockout mice. All results represent data generated in at least three independent experiments. Error bars mean ⁇ SD. Data were analyzed using one-way ANOVA with Bonferroni correction or independent unpaired two-tailed Student's t-test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG. 13(13A) Compared with the group of BMMSCs implanted under the renal capsule alone, the blood glucose of the mice in the group injected with the cytokine IL-6 by intraperitoneal injection of BMMSCs under the renal capsule was more significantly reduced. Diabetes was induced in wild-type mice with multiple injections of low-dose streptozotocin (STZ, five doses of 50 mg/kg body weight). The mice in the BMMSC+IL-6 group were given intraperitoneal injection of IL-6 (2ng/ml, 200 ⁇ l per mouse) for four consecutive times on the day of transplantation of MSCs and every week thereafter, except for BMMSCs implanted under the renal capsule. .
  • STZ low-dose streptozotocin
  • mice in the group of BMMSCs implanted under the renal capsule and injected with the cytokine IL-6 by intraperitoneal injection had stronger glucose clearance ability.
  • pancreatic exocrine gland-derived mesenchymal stem cells express multiple markers of pancreatic progenitors, including PDX1, Nkx6.1 and Ngn3, they predominantly regenerate the pancreas when implanted in the renal capsule Exocrine glands (soil), with very few insulin-secreting cells (seeds) in the regenerating tissue.
  • PMSCs pancreatic exocrine gland-derived mesenchymal stem cells
  • PMSCs improved the pancreatic immune microenvironment (soil) by expressing high levels of IL-6 to inhibit IL-17, which in turn rescued damaged islet ⁇ cells (seeds). Furthermore, we found that renal capsule implantation of PMSCs showed a significant and sustained therapeutic effect in STZ-induced mice compared with intravenous injection of PMSCs. PMSC-mediated regeneration of exocrine pancreas rescues pancreatic islet ⁇ -cell damage in type 1 diabetic mice. This study sheds light on the important role of exocrine pancreatic regeneration in protecting beta cells and provides a new "soil rescue seed" strategy for type 1 diabetes treatment.
  • STZ means streptozotocin.
  • pancreatic lipase are specific markers of pancreatic exocrine glands, and the "insulin” is a pancreatic islet beta cell marker.
  • the acinar and ductal cell markers such as amylase, trypsin, pancreatic lipase and CK19, etc.
  • composition may comprise A alone; B alone; C alone; D alone ; Combinations containing A and B; Combinations containing A and C; Combinations containing A and D; Combinations containing B and C; Combinations containing B and D; Combinations containing C and D; Combinations containing A, B and C Combination; A, B, and D combination; A, C, and D combination; B, C, and D combination; or A, B, C, and D combination used.
  • the components in the "composition” may be present in a mixed form, or may be packaged separately.
  • the separately packaged components may also contain their respective adjuvants.
  • the adjuvant refers to a means that can assist the curative effect of a drug in pharmacy.
  • the separate packaged components may be administered simultaneously or in any sequential order wherein the patient is first treated with one drug and then administered the other drug .
  • the patient refers to a mammalian subject, especially a human.
  • treatment refers to an effect that occurs when a patient suffers from a particular disease, disease or condition, which reduces the severity of the disease, condition or condition, or delays or slows the disease.
  • a disorder or disorder (“therapeutic treatment”), and also contemplates actions that occur before a patient begins to suffer from a particular disease, disorder or disorder (“prophylactic treatment”).
  • mice Female C57BL/6J, NU/j, NOD/SCID, IL-6 knockout mice were used in this experiment. Age-matched 8- to 10-week-old female mice of the same background were used in all experiments. All animal experiments were performed according to institutional approved protocols for animal research (SYSU-IACUC #2020000122 of Sun Yat-Sen University and IACUC #805478 of University of Pennsylvania). Mice were euthanized following MSC implantation at the times indicated for each experiment. To destroy endogenous islet cells to induce type 1 diabetes, intraperitoneal (ip) injection of streptozotocin (STZ) with 0.01 M Sodium citrate dissolves STZ at pH 4.5 and is injected into 8 week old mice.
  • ip intraperitoneal injection of streptozotocin
  • STZ streptozotocin
  • mice blood samples from the tail vein of mice were taken to measure circulating glucose levels. Two consecutive measurements of non-fasting blood glucose ⁇ 300 mg/dl were considered to be indicative of significant diabetes features (77).
  • IPGTT intraperitoneal glucose tolerance test
  • animals were fasted for 5 hours and then injected intraperitoneally. 20% glucose solution (2 g glucose per kg body weight) was injected. Blood glucose was measured at 0, 30, 60, 90 and 120 minutes after injection.
  • splenocytes were harvested and stained with anti-CD4-PerCP and CD25-APC antibodies for 30 min on ice. Next, cells were stained with anti-Foxp3-PE antibody using Foxp3 staining buffer kit (eBioscience, San Diego, CA, USA) for cell fixation and permeabilization.
  • cytokine staining such as IL-17A
  • splenocytes were treated with 500 ng/ml PMA and 500 ng/ml ionomycin (Peprotech, Rocky Hill, NJ, USA) for 5 hours, and Brefeldin A (BFA; 5 ⁇ g/ml) was added for 5 hours. ml, Peprotech), within the last 4 hours of culture.
  • cells were stained with anti-CD4-PerCP antibody followed by anti-IL17A PE antibody using a staining buffer kit. All antibodies were purchased from BioLegend (San Diego, CA, USA). Samples were analyzed with a FACScalibur flow cytometer (BD Bioscience, San Jose, CA, USA).
  • cryosection samples dissected pancreata were fixed in 4% PFA at 4°C for 4 hours, washed 3 times with PBS, and then embedded in optimal cutting temperature (OCT) compound. Tissue sections were incubated with primary antibodies overnight at 4°C, then washed in PBST (PBS + 0.1% Triton X-100), incubated with secondary antibodies and DAPI for 2 hours at room temperature, then washed and fixed for confocal imaging . For whole-wall staining, islets were fixed in 4% PFA at 4 °C for 1 h, washed 3 times with PBS, and then blocked with whole-wall blocking buffer for 1 h at room temperature. Blocking buffer contains 10% FBS in PBST. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C, followed by 3 washes with PBST, and then secondary antibodies and DAPI were incubated overnight at 4°C. Photographs were taken using a laser confocal microscope.
  • mice Serum and tissue samples from mice were measured for each cytokine and C-peptide levels using ELISA kits purchased from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions.
  • Protein extraction reagent (Thermo, Waltham, MA, USA) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) and protein concentration assays (Bio-Rad Laboratories, Hercules, CA USA) were used ) to measure the protein concentration. 20 ⁇ g of protein were separated by vertical electrophoresis using SDS-PAGE gels and transferred to 0.2 ⁇ m nitrocellulose membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% nonfat dry milk and 0.1% Tween-20 for 1 hour, then primary antibodies were diluted in blocking solution overnight according to the manufacturer's instructions.
  • Membranes were then incubated in HRP-conjugated secondary antibody (Santa Cruz) diluted 1:10,000 in blocking buffer for 1 hour at room temperature. Protein expression was detected using SuperSignal West Pico chemiluminescent substrate (Thermo) and a Bio-Rad ChemiDoc TM Imager (Hercules, CA, USA).
  • RNA samples (1 ⁇ g) were reverse transcribed in a reverse transcription system (QIAGEN).
  • the primers used in PCR experiments were: mouse IL-6 upstream primer GGCGGATCGGATGTTGTGAT, and downstream primer GGACCCCAGACAATCGGTTG. PCR conditions were: 95°C for 5 minutes, (95°C for 10 seconds, 50°C for 45 seconds) ⁇ 40 and 90°C for 10 seconds.
  • CD4+ T lymphocytes were isolated from splenocytes using BD IMag TM anti-mouse CD4Particles-DM (BD Bioscience) according to the manufacturer's instructions.
  • CD4+ T cells were prestimulated for 2 days with plates conjugated with anti-CD3e antibody (clone 145-2C11, 5 ⁇ g/ml, BioLegend) and soluble anti-CD28 antibody (clone 37.51, 5 ⁇ g/ml, BioLegend).
  • T helper cells 17 activated T cells (1 ⁇ 10 6 ) were added to 0.2 ⁇ of recombinant human TGF- ⁇ 1 (2 ⁇ ng/ml; R&D Systems, Minneapolis, MN, USA) 106 MSCs mouse IL-6 (25 ⁇ ng/ml; Peprotech), recombinant mouse IL-23 (20 ⁇ ng/ml; Peprotech) and recombinant mouse IL- ⁇ 1 (20 ng/ml; Peprotech) (89). After 3 days, cells in suspension were harvested for IL-17A antibody staining and analyzed with FACScalibur (BD Bioscience).
  • Example 1 Isolation, culture and identification of pancreatic mesenchymal stem cells (PMSCs)
  • the steps of isolation and culture include:
  • Pancreatic tissue from mice was gently dissociated, minced, and treated with phosphate containing 2 mg/mL collagenase type I (Worthington Biochemical, Lakewood, NJ, USA) and 4 mg/mL Dispase II (Roche Diagnostics, Basel, Switzerland) Buffer (PBS) for 1 hour at 37°C for digestion.
  • PBS Buffer
  • a single cell suspension of the pancreas was obtained by passing the digested cell suspension through a 70 ⁇ m filter (BD Biosciences, San Jose, CA, USA).
  • ANC nucleated cells
  • PMSCs were further characterized and demonstrated that PMSCs have the ability of self-renewal (assessed by CFU-F) and high proliferation rate (assessed by Brdu staining):
  • pancreatic tissue-derived CD105 + PMSCs co-express ⁇ cell marker insulin (insulin), acinar cell marker amylase (acinar cell marker amylase), and ductal cell marker (pancreatic progenitor cell markers) ) CK19, pancreatic progenitor cell markers Ngn 3, Ptf1a, Hnf1 ⁇ , PDX1 and Nkx6.1 ( Figure 1A).
  • pancreatic tissue-derived MSCs positively expressed the mesenchymal stem cell surface markers CD44, CD105, CD73 and Sca-1, but not the blood cell markers CD34 and CD45 (Fig. 1B).
  • adipogenic induction and staining were also carried out according to the experimental standard procedures of commercial kits.
  • the experimental methods for CFU-F evaluation are as follows: 1) 5 ⁇ 10 3 mononuclear cells (ANCs) isolated from the pancreas were seeded in 60 mm culture plates;
  • the steps of the BrdU labeling method are as follows: 1) The cells were seeded in a 12-well plate with a cell number of 1 ⁇ 10 5 /mL (a cover glass was placed inside), cultured for 1 day, and synchronized with a medium containing 0.4% FBS for 3 days. The vast majority of cells are in G0 phase;
  • PMSC has stronger CFU-F formation and proliferation ability, stronger osteogenic differentiation ability and lower adipogenic differentiation ability. Similar to BMMSCs, PMSCs expressed MSC-positive surface markers Sca-1, CD105, CD44, CD73 but not blood cell surface markers CD34 and CD45.
  • PMSCs pancreatic exocrine gland-derived MSCs
  • mature islet-specific markers including insulin, as well as mature exocrine gland markers amylase and ductal cell marker cytokeratin 19 (CK19).
  • Fig. 1F pancreatic progenitor markers PDX1, Nkx6.1, as well as endocrine differentiation marker Ngn 3 and acinar differentiation marker Ptf1a by flow cytometry analysis (Fig. 1A, 1G) and immunofluorescence staining (S1B). and the ductal differentiation marker Hnf1 ⁇ .
  • PMSCs possess general MSC properties, including the expression of MSC surface molecules and the capacity for self-renewal and pluripotent differentiation.
  • Our assay results showed that PMSCs had clonogenic (CFU-F) ability and higher proliferation rate compared with bone marrow MSCs (BMMSCs) (Fig. 1E).
  • CFU-F clonogenic
  • BMMSCs bone marrow MSCs
  • Flow cytometry analysis confirmed that PMSCs positively expressed mesenchymal stem cell surface markers CD44, CD73, CD105 and Sca-1, and negatively expressed blood cell markers CD34 and CD45 (Fig. 1B).
  • PMSCs also showed the ability to differentiate into pluripotent, including osteogenic, adipogenic differentiation (Fig. 1C, 1D).
  • Culture and expand pancreatic mesenchymal stem cells in a 10cm cell culture dish routinely change the medium until the cells are in contact with fusion, discard the culture medium, digest into single cells, and collect about 4 ⁇ 10 6 mesenchymal cells; use about 20 ⁇ L 1 Suspend cells in ⁇ PBS solution and put them on ice for later use; or mix the above PBS cell suspension with a small piece of absorbable gelatin sponge, and place the mixture on ice for later use.
  • the adherent PMSCs were completely peeled from the petri dish, and then transferred to a new 24-well plate for continued cultivation. After 1-3 days, PMSC pellets were formed, and the PMSC pellets were placed on ice for use. (There was no significant difference in regeneration results after implantation under the renal capsule in several ways.)
  • Surgical scissors make a longitudinal incision of about 2 cm in the skin to expose the kidney. Use the tips of microsurgical scissors or micro forceps to make an opening under the renal capsule and bluntly separate the renal capsule from the submembranous renal tissue to make a pocket for the implant.
  • the PMSC+gelatin sponge mixture or PMSC globules were placed in the subrenal capsule, or the PMSC cell suspension was injected into the subrenal capsule.
  • the renal capsule wound was closed with an electrocautery, the kidney was put back in place, and the surgical wound was sutured. After 8 weeks, the grafts were collected and tested accordingly.
  • PMSCs When PMSCs were implanted under the renal capsule of wild-type or immunodeficient mice, they ectopically regenerated pancreatic exocrine tissue without endocrine islets 8 weeks after implantation (Fig. 2A-D). Similar to normal pancreatic exocrine glands, the regenerated exocrine pancreas consisted of typical acini and ducts (Fig. 2C-E,). The regenerated acini displayed exocrine pancreatic cells with strongly basophilic staining properties with eosinophilic secretory granules and ducts with epithelioid linings ( Figure 2C-E).
  • Example 4 PMSC-mediated regeneration of subrenal capsular pancreas ameliorates STZ-induced type I diabetes
  • Example 5 Subrenal implantation of PMSCs rescues damaged orthotopic pancreas/islets in type 1 diabetic mice
  • Fig. 8A Immunostaining analysis showed that IL-6 was expressed in both pancreatic islets and exocrine pancreas (Fig. 8B). Furthermore, we found that PMSCs, but not BMMSCs, protected the pancreatic ⁇ cell line MIN6 from STZ-induced apoptosis in an in vitro co-culture system (Fig. 8C, 8D).
  • Example 7 Subrenal implantation of PMSCs increases the level of IL-6
  • Example 8 IL-6 knockout mice show resistance to PMSC implantation therapy
  • Example 9 The role of IL-6 in the treatment of type 1 diabetes by transplantation of bone marrow mesenchymal stem cells under the renal capsule
  • mice in the bone marrow mesenchymal stem cell (BMMSC)+IL-6 group were transplanted on the day of BMMSC transplantation and every subsequent day. Weeks, four consecutive intraperitoneal injections of IL-6 (2 ng/mL, 200 ⁇ L per injection per mouse) were administered.
  • IPGTT glucose tolerance test

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Abstract

提供了一种来源于胰腺外分泌腺的间充质干细胞或胰腺外分泌腺组织在制备治疗/预防糖尿病药物当中的应用，所述胰腺外分泌腺通过胰腺外分泌腺来源的间充质干细胞异位再生自然分化形成，将胰腺间充质干细胞异位移植入动物体内，通过再生胰腺外分泌腺达到治疗糖尿病的作用。

Description

一种治疗糖尿病的药物及其方法 技术领域
本公开属于生物医药领域，涉及一种治疗糖尿病的药物及其方法。
背景技术
糖尿病是由胰岛素不足及其功能丧失而发展成的疾病，一旦发展成糖尿病很难治愈。糖尿病可主要分为I型糖尿病(胰岛素依赖型糖尿病)和II型糖尿病(非胰岛素依赖型糖尿病)两种类型。II型糖尿病是由胰岛素抗性发展成的慢性疾病，它成为与生活习惯有关的问题，例如由于过度进食和不活动引起的肥胖，压力等，II型糖尿病通常在中老年人中发展，并且许多糖尿病患者患有II型糖尿病。I型糖尿病是由自身免疫性疾病，病毒感染等破坏胰腺细胞(也称作胰岛素产生细胞)以终止体内胰岛素分泌而引起的慢性疾病。
近年来，糖尿病发病率呈上升趋势，而且趋于年轻化，糖尿病已成为严重危害人类健康的常见内分泌代谢性疾病。不同类型的糖尿病都会导致胰腺内的β细胞不能产生足量的胰岛素以降低血糖的浓度，导致高血糖症的发生。目前治疗的手段主要采用药物治疗，药物治疗虽能降低血糖，无法对糖尿病进行根治；使用胰岛素进行治疗可以控制症状，但是长期使用会产生对胰岛素的耐受。胰岛移植手术风险小，是治愈糖尿病的有效手段，但供体相对不足及移植后的免疫排斥反应等严重阻碍了该疗法的广泛应用。糖尿病由于其广泛的流行性以及缺乏可治愈的方法而成为主要的公共卫生问题。
发明内容
一些实施方案中，本公开提供了来源于胰腺外分泌腺的间充质干细胞或胰腺外分泌腺组织在制备治疗/预防糖尿病药物当中的应用。
一些实施方案中，所述的胰腺外分泌腺组织由胰腺外分泌腺的间充质干细胞生成。
一些实施方案中，所述的胰腺外分泌腺的间充质干细胞药物为注射制剂。
一些实施方案中，所述的胰腺外分泌腺的间充质干细胞药物为肾囊膜下注射制剂。
一些实施方案中，本公开提供了促进形成胰腺外分泌腺的物质在制备治疗/预防糖尿病药物当中的应用，所述胰腺外分泌腺通过胰腺外分泌腺来源的间充质干细胞异位再生自然分化形成。
胰腺具有内分泌腺(内分泌细胞)和外分泌腺(外分泌物细胞)，并且是在这两种分泌细胞中起重要作用的器官。外分泌物细胞主要起到分泌胰脂肪酶，胰蛋白酶，弹性蛋白酶，胰淀粉酶等消化酶的作用。
一些实施方案中，所述糖尿病为Ⅰ型糖尿病。
一些实施方案中，本公开提供了一种用于治疗治疗/预防糖尿病的系统，包含胰腺外分泌腺的间充质干细胞，以及植入装置。一些实施方案中，所述的植入装置用于将胰腺外分泌腺的间充质干细胞植入患者体内定点部位。
一些实施方案中，所述的介入装置为穿刺器。
一些实施方案中，所述的定点部位为包括肾囊膜下、皮下、或肠系膜。
一些实施方案中，所述的系统还包含超声装置或内窥镜装置，所述超声装置或内窥镜装置用于引导所述穿刺器的定位。
一些实施方案中，所述植入为肾囊膜下植入。
一些实施方案中，所述异位再生的位置包括肾囊膜下、皮下。
其中，Ⅰ型糖尿病患者体内胰岛β细胞坏死，不产生胰岛素，目前无治愈手段。近来国内外均有研究报道MSC可促进胰岛细胞再生，MSC移植有望成为糖尿病治疗的新方法，尤其是针对1型糖尿病的治疗。也就是说，目前普遍都认为间充质干细胞通过再生胰岛β细胞达到治疗糖尿病的作用。例如2008年，同济大学附属第十人民医院宋振顺团队发现胰腺干细胞可分化为分泌胰岛素的胰岛样结构，胰岛及胰腺干细胞来源的胰岛样结构序贯移植对大鼠糖尿病有治疗作用。他们还发现具有多向分化潜能的MSC在体内也可分化为产胰岛素细胞(insulin-producing cell，IPC)和血管内皮细胞，MSC及其来源的IPC移植明显促进了受体残余胰岛周围新生血管的生成，进而使残余胰岛得到增殖，这一研究得到Barky等的证实。此外，Arzouni等的研究也发现MSC移植后可通过释放膜联蛋白A1(ANXA1)和细胞外基质改善人胰岛功能。一些研究还表明，自体来源的MSC和胰岛共同移植可提高胰岛移植的成功率和安全性，有效减轻异体移植免疫排斥反应及早期因炎症反应引起的移植物损失，或可为1型糖尿病治疗提供新的方案。再例如Daisong Wang等的研究提到，胰岛祖细胞可形成胰岛样组织，其中胰岛β细胞在组织中占主要的。这种组织长期培养可以逆转糖尿病。
可见，现有技术普遍认为，细胞治疗糖尿病主要是因为再生了胰岛细胞。本领域技术人员在研究糖尿病的时候，也普遍地将胰岛细胞作为研究目标。
而发明人克服本领域的技术偏见，惊奇地发现，将胰腺间充质干细胞异位移植入动物体内，通过再生胰腺外分泌腺而不是胰岛细胞达到治 疗糖尿病的作用，并且其治疗非常显著。
在一些实施例中，发明人研究发现，PMSCs通过抑制IL-17来表达高水平的IL-6，以改善胰腺免疫微环境，从而挽救受损的胰岛细胞。此外，我们使用基因敲除小鼠模型显示缺乏IL-6会导致STZ诱导的1型糖尿病的严重性增加，并且对PMSC植入的治疗有抵抗力，从而证实了PMSC作用于IL-6保护β细胞。
一些实施方案中，本公开还提供了一种获得胰腺外分泌腺的组织的方法，所述的胰腺外分腺的组织通过植入间充质干细胞而形成。
在一些实施方案中，所述的胰腺外分腺的组织通过向动物体内异位植入间充质干细胞而形成。在一些实施方案中，所述动物选自猪、大鼠、小鼠、仓鼠、兔、猪、牛、鹿、绵羊、山羊、小鸡、猫、马、狗、猩猩、猴。在一些实施方案中，所述动物选自小鼠。
在一些实施方案中，所述植入包括肾囊膜下植入、皮下植入、肠系膜植入等。
在一些实施方案中，所述植入为肾囊膜下植入。
在另一些实施例中，所述的胰腺外分腺的组织以非活体的组织模型植入。
在一些实施方案中，所述间充质干细胞包括胰腺间充质干细胞、骨髓间充质干细胞。
在一些实施方案中，所述间充质干细胞为胰腺间充质干细胞。
在一些实施方案中，发明人研究发现，与静脉内输注PMSCs相比，PMSCs的肾囊膜下植入在STZ诱导的小鼠中显示出显著的持续治疗效果。本公开揭示了外分泌胰腺再生在保护β细胞方面的未知作用，并证明了1型糖尿病治疗的土壤拯救种子策略。
在一些实施方案中，所述的间充质干细胞为游离的间充质干细胞。
在一些实施方案中，所述的间充质干细胞无需处理直接植入。而在目前的现有技术中，用间充质干细胞治疗糖尿病，间充质干细胞一般都会先在体外进行处理激活。例如BEHROUS DAVANI等人研究人胰岛来源的间充质干细胞用于治疗I型糖尿病时，从胰岛取得细胞后，使用生长培养基进行体外扩增，向扩增后的间充质干细胞中加入分化诱导液进行诱导(诱导液成分如下：CMRL-1066培养液加入2mM L-glutamine,1％(wt/vol)bovine serum albumin(BSA)，Fraction V Fatty Acid Free(MP Biomedicals,Irvine,CA,http://www.mpbio.com),and 1×insulin-transferrinselenium-A(Gibco))。诱导4天后，间充质干细胞形成了上皮细胞簇，将上皮细胞簇移通过肾囊膜下植入后，形成了胰岛样的细胞聚集体(BEHROUS DAVANI，et al.Human Islet-Derived Precursor Cells Are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone-Expressing Cells In Vivo.Stem Cells,2007,25:3215–3222.)。再例如Daisong Wang等通过流式分选Procr +胰腺干细胞与内皮细胞在体外共培养、扩增一段时间后植入体内，形成胰岛组织，移植小鼠体内，逆转糖尿病(Daisong Wang，et al.Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+Progenitors.Cell,2020,180:1198–1211.)。
而本公开则可以直接将体外常规培养的间充质干细胞，消化成单个游离的细胞之后，直接植入动物体内即可达到治疗效果，具有非常好的可操作性，易于推广使用。
一些实施方案中，所述的间充质干细胞可以与明胶海绵等支架材料混合。
在一些实施例中，所述的培养扩增的胰腺来源的间充质干细胞(PMSC)表达了多种胰腺祖细胞标记PDX1，Nkx6.1，Ptf1a，Hnf1b和Ngn3。
一些实施方案中，本公开提供了一种治疗糖尿病的方法，所述治疗糖尿病的方法包括以下步骤：(1)用培养液培养间充质干细胞；(2)去掉步骤(1)中的间充质干细胞的培养液，收集间充质细胞；(3)将步骤(2)中收集到的间充质干细胞用PBS或生理盐水(但又不限于此)混悬后，植入动物肾囊膜下、皮下或肠系膜上，从而达到治疗效果。
在一些实施方案中，所述获得胰腺外分泌腺的组织的方法包括以下步骤：(1)用培养液培养间充质干细胞；(2)去掉步骤(1)中的间充质干细胞的培养液，收集间充质细胞；(3)将步骤(2)中收集到的间充质干细胞用PBS或生理盐水(但又不限于此)混悬后，植入动物肾囊膜下、皮下或肠系膜上，从而获得胰腺外分泌组织。
在一些实施方案中，所述PBS为微量的PBS。
一些实施方案中，所述的间充质干细胞可以与明胶海绵等支架材料混合。
在一些实施方案中，步骤(1)中的培养液的成分包括FBS，谷氨酰胺(200mM)，2-ME和α-MEM。
在一些实施方案中，步骤(1)中的培养液的成分包括FBS，青霉素/链霉素溶液，谷氨酰胺(200mM)，2-ME(55mM)，α-MEM。
在一些实施方案中，所述间充质干细胞培养至P1-P3代，但又不限于此。
在一些实施方案中，本公开提供了一种胰腺外分泌腺的组织，所述的胰腺外分泌腺的组织通过所述的方法获得。
在一些实施方案中，本公开提供了所述的胰腺外分泌腺的组织在胰腺相关疾病的治疗药物的发现筛选、或胰腺相关疾病治疗药物的毒性测定、或胰腺胚胎学、或胰腺细胞谱系和分化途径的研究、或基因表达研究、或参与胰腺损伤和修复的机制研究、或胰腺的炎症性疾病研究、或治病机制研究中的体外用途。
在一些实施方案中，所述的胰腺外分泌腺的组织在重组基因表达中的应用。
在一些实施方案中，所述的胰腺外分泌腺的组织在制备用于治疗胰腺病症或胰腺疾病的药物中的应用，或者在制备用于再生医学的药物中的用途。
在一些实施方案中，所述的胰腺疾病包括糖尿病。
在一些实施方案中，所述的糖尿病优选为Ⅰ型糖尿病。
在一些实施方案中，含有IL-6和间充质干细胞的组合物在制备治疗和/或预防糖尿病药物方面的应用。
在一些实施方案中，所述间充质干细胞的来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱、外周血或胰腺。
在一些实施方案中，所述间充质干细胞的来源为骨髓。
在一些实施方案中，所述骨髓间充质干细胞为游离的骨髓间充质干细胞。
在一些实施方案中，所述IL-6和间充质干细胞被同时给药。
在一些实施方案中，所述IL-6和间充质干细胞被单独给药。
在一些实施方案中，先给予IL-6，后给予间充质干细胞。
在一些实施方案中，先给予间充质干细胞，后给予IL-6。
在一些实施方案中，所述药物为注射制剂。
在一些实施方案中，所述药物选自静脉注射剂、肌肉注射剂、皮下注射剂或鞘内注射剂。
在一些实施方案中，所述间充质干细胞为肾囊膜下注射剂。
在一些实施方案中，所述IL-6为腹腔注射剂。
在一些实施方案中，所述药物还包括药学上可接受的药用载体。
在一些实施方案中，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
在一些实施方案中，所述糖尿病为Ⅰ型糖尿病。
发明人研究发现，骨髓间充质干细胞治疗Ⅰ型糖尿病的效果远远没有胰腺外分泌腺来源的间充质干细胞好，且骨髓间充质干细胞与胰岛β细胞共培养(直接接触)，骨髓间充质干细胞对胰岛β细胞有杀伤作用。这导致了骨髓间充质干细胞不是治疗Ⅰ型糖尿病的理想细胞来源。但是发明人没有局限于这种现状，而是经过不断探索研究发现，向骨髓间充质干细胞添加IL-6后，意外发现可以大大提高治疗Ⅰ型糖尿病的效果。从而克服了骨髓间充质干细胞治疗Ⅰ型糖尿病效果不佳的技术障碍，扩大了治疗Ⅰ型糖尿病的细胞来源。使得骨髓间充质干细胞这种相对于胰腺外分泌腺来源的间充质干细胞更易获得的，并且来源广泛的细胞可以用于临床上。具有非常好的现实意义。
在一些实施方案中，本公开提供了一种用于治疗/预防糖尿病的系统，包含间充质干细胞和IL-6，以及植入装置。
在一些实施方案中，所述的植入装置用于将间充质干细胞植入患者体内定点部位。
在一些实施方案中，所述的植入装置为穿刺器。
在一些实施方案中，所述定点部位包括肾囊膜下、皮下、或肠系膜。
在一些实施方案中，所述的系统还包含超声装置或内窥镜装置。
在一些实施方案中，所述超声装置或内窥镜装置用于引导所述植入装置的定位。
在一些实施方案中，本公开提供了一种治疗糖尿病的方法，包括给予患者来源于胰腺外分泌腺的间充质干细胞、或移植所述的胰腺外分泌腺组织、或给予含有IL-6和间充质干细胞的组合物。
在一些实施方案中，所述糖尿病为Ⅰ型糖尿病。
在一些实施方案中，所述的胰腺外分泌腺的间充质干细胞通过肾囊膜下植入、皮下植入、肠系膜植入给予所述的患者。
在一些实施方案中，所述植入为异位植入。
在一些实施方案中，所述异位植入为非活体的组织模型植入。
在一些实施方案中，所述IL-6和间充质干细胞的组合物中，所述间充质干细胞的来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱、外周血或胰腺。
在一些实施方案中，所述骨髓间充质干细胞为游离的骨髓间充质干细胞。
在一些实施方案中，所述IL-6和间充质干细胞被同时给药。
在一些实施方案中，所述IL-6和间充质干细胞被单独给药。
在一些实施方案中，先给予IL-6，后给予间充质干细胞。
在一些实施方案中，先给予间充质干细胞，后给予IL-6。
在一些实施方案中，所述组合物为注射制剂。
在一些实施方案中，所述组合物选自静脉注射剂、肌肉注射剂、皮下注射剂或鞘内注射剂。
在一些实施方案中，所述组合物中，所述间充质干细胞为肾囊膜下注射剂。
在一些实施方案中，所述组合物中，所述IL-6为腹腔注射剂。
在一些实施方案中，所述组合物还包括药学上可接受的药用载体。
在一些实施方案中，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
附图说明
图1A-1G为鼠PMSC的鉴定。(1A)流式细胞仪分析证实CD105 +PMSCs共表达β细胞标记物胰岛素，腺泡细胞标记物淀粉酶，导管细胞标记物CK19和胰腺祖细胞标记物Ngn 3，Ptf1a，Hnf1β，PDX1和Nkx6.1。(1B)流式细胞仪分析显示，PMSC表达间充质干细胞表面标志物Sca-1，CD105，CD73，CD44，而血液细胞标志物CD34和CD45呈阴性。(1C,1D)PMSCs具有成骨和成脂分化潜能,n＝3。(1E)PMSCs显示出形成CFU-F的能力，并且比BMMSCs具有更高的增殖速率,n＝3。(1F)免疫荧光染色显示CD105 +PMSCs表达胰岛细胞标志物胰岛素(insulin)，胰腺外分泌腺细胞标志物淀粉酶(amylase)，导管细胞标记物CK19，胰腺祖细胞标记物Ngn3，Ptf1a，Hnf1β，PDX1和Nkx6.1。(1G)流式细胞仪分析表明PMSCs表达了间充质干细胞表面标记CD44，并共表达β细胞标记胰岛素，腺泡细胞标记淀粉酶和胰腺祖细胞标记Ngn 3，Ptf1a，Hnf1β，PDX1和Nkx6.1。
图2A-2F为胰腺干细胞再生异位外分泌胰腺。(2A)野生型小鼠异位再生胰腺的H&E染色和免疫荧光染色。(2B)代表性H&E和免疫荧光图像显示，野生型小鼠中肾囊膜下植入的PMSCs异位再生外分泌胰腺，其含有数量有限的胰岛素阳性细胞。比例尺，500μm。(2C，2D)代表性H&E和免疫荧光图像显示，免疫缺陷小鼠中肾囊膜下植入的PMSCs异位再生外分泌胰腺，其含有数量有限的胰岛素阳性细胞。免疫荧光显示异位再生胰腺中胰岛素和淀粉酶均呈双重染色。右图是左H&E图像中来自方框区域的免疫染色图像的更高放大倍数视图。黑色虚线表示肾脏和再生的外分泌胰腺的交界处。黄色虚线表示外分泌胰腺腺泡。Kidney：肾，EP：异位胰腺。(2E)野生型小鼠的正常和异位再生胰腺的H&E和免疫荧光染色。异位再生胰腺的H&E染色显示外分泌胰腺区室的典型染色，与正常胰腺相似。免疫荧光检测显示，异位再生的胰腺其内分泌细胞标志物，例如β细胞的胰岛素、α细胞的胰高血糖素、δ细胞的生长抑素和PP细胞的胰腺胰多肽染色均呈阴性；但腺泡和导管细胞标志物如淀粉酶、胰蛋白酶、胰脂酶和CK19染色呈阳性。右图是左H&E和免疫染色图像中方框区域的较高放大率视图。白色虚线表示肾脏与再生的外分泌胰腺的交界处。黄色虚线表示外分泌胰腺腺泡。黑色虚线表示导管。EP：异位胰腺。比例尺，低放大倍数为200μm，高放大倍数为50μm。(2F)定量分析表明植入的80.0％PMSCs在肾囊膜下再生了外分泌胰腺；在再生的外分泌胰腺中，仅检测到12.5％的胰岛素阳性细胞。所有结果是代表至少三个独立实验中产生的数据。
图3A-3M为PMSCs介导的肾囊膜下胰腺再生改善了STZ诱导的I型糖尿病。(3A)与BMMSC植入组相比，肾囊膜下植入PMSCs可以显著降低1型糖尿病小鼠的血糖水平。用多次低剂量链脲佐菌素(STZ，五剂50mg/kg体重)注射液诱导野生型小鼠糖尿病。使用尾静脉血液样本(n＝5)测量指定组小鼠的血糖水平。(3B)PMSC植入挽救了葡萄糖清除能力。植入后2个月使用腹腔注射葡萄糖耐量实验(IPGTT)检测小鼠葡萄糖清除能力，n＝3。(3C,3D)PMSC植入增强了血清C肽和胰岛素水平。其中植入后2个月通过ELISA测量血液C肽和胰岛素水平，n＝5。(3E)代表性的H&E和免疫荧光图像显示了在1型糖尿病小鼠中，PMSCs的肾囊膜下植入再生了异位外分泌胰腺，此再生胰腺中检测不到胰岛素阳性的细胞。(3F)植入PMSCs显著提高了免疫缺陷小鼠单次高剂量注射STZ(200mg/kg体重)诱导的1型糖尿病的存活率。糖尿病小鼠的Kaplan-Meier生存分析表明，与BMMSC植入相比，PMSC植入显著改善了小鼠的寿命(STZ和BMMSC-KC组为n＝4，PMSC-KC组为n＝5)。(3G)PMSC植入可显著降低1型糖尿病小鼠的血糖水平。通过收集尾静脉血样植入来测量指定组中每只小鼠的血糖水平(STZ和BMMSC-KC组为n＝4，PMSC-KC组为n＝5)。(3H)PMSC植入挽救了葡萄糖清除能力。植入后2个月使用腹腔注射葡萄糖耐量实验(IPGTT)检测小鼠葡萄糖清除能力，n＝3。(3I)具有代表性的H&E和免疫荧光图像显示，在1型糖尿病小鼠中，PMSCs的肾囊膜下植入可以再生无法检测到产胰岛素细胞的异位外分泌胰腺。KC：MSCs的肾囊膜下植入。(3J-3L)与全身输注PMSCs相比，PMSCs的肾脏囊膜下植入在葡萄糖清除率，血清C肽和胰岛素水平方面获得了更大的改善。在植入后2个月，在IPGTT测试期间测量血糖水平，n＝3。在植入后2个月，通过ELISA测量血液C肽水平，n＝5。(3M)与通过尾静脉全身注入PMSCs相比，PMSCs的肾囊膜下植入对1型糖尿病小鼠产生了长期的血糖改善。使用尾静脉血液样本(n＝5)测量指定组小鼠的血糖水平。PMSC-systemic：PMSC的全身性输注。比例尺，50μm。所有结果代表至少三个独立实验中产生的数据。误差线＝平均值±SD。使用带有Dunnett检验的双向ANOVA(two-way ANOVA)或带有Bonferroni校正的单向ANOVA(one-way ANOVA)对数据进行分析，以比较多个组。在一个组内，将每个时间点与时间＝0组进行比较；在组之间，将每个组与STZ组进行比较。NS，无统计学差异。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
图4A-4F为PMSCs的肾囊膜下植入可挽救1型糖尿病小鼠受损的原位胰腺/胰岛。(4A)接受了PMSCs的肾囊膜下植入的1型糖尿病小鼠的原位胰腺/胰岛的代表性H&E图像。黑色虚线表示1型糖尿病小鼠胰腺中的胰岛。(4B)免疫荧光染色显示，来自1型糖尿病小鼠的胰腺中的胰岛素和淀粉酶共染色。黑色虚线表示1型糖尿病小鼠的胰岛。(4C)Ki67和胰岛素共同染色显示，植入PMSCs显著增加了1型糖尿病小鼠受损的胰岛β细胞的增殖。(4D)代表性的H&E图像显示，与全身输注PMSCs相比，PMSCs的肾囊膜下植入可大大挽救1型糖尿病小鼠的胰岛受损。右图显示了胰岛面积的定量，n＝5。(4E)免疫荧光染色显示1型糖尿病小鼠胰腺中胰岛素和淀粉酶共染色。黑色虚线表示1型糖尿病小鼠胰腺中的胰岛。右图显示了胰岛素阳性细胞的定量，n＝5。(4F)Ki67和胰岛素共染色显示，与全身性输注PMSCs相比，PMSCs可显著增强1型糖尿病小鼠胰岛β细胞受损的增殖。右图显示Ki67和胰岛素双阳性细胞的定量，n＝5。PMSC-KC：PMSCs的肾囊膜下植入，PMSC-systemic：PMSCs的系统性输注。所有结果代表至少三个独立实验 中产生的数据。使用带有Dunnett检验的双向ANOVA(two-way ANOVA)或带有Bonferroni校正的单向ANOVA(one-way ANOVA)对数据进行分析，以比较多个组。在一个组内，将每个时间点与时间＝0组进行比较；在组之间，将每个组与STZ组(a，b，f，g)进行比较。NS，无统计学差异。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
图5A-5B为胰腺外分泌腺特异性标志物胰蛋白酶和胰脂肪酶与β细胞标志物胰岛素的共染色。(5A，5B)免疫荧光染色显示1型糖尿病小鼠胰腺中的胰岛素与腺泡细胞标志物胰蛋白酶和胰脂酶共染色。与BMMSCs植入或全身输注PMSCs相比，PMSCs的肾囊膜下植入可显著挽救STZ诱导的受损的胰岛素阳性β细胞以及胰蛋白酶和胰脂肪酶阳性的腺泡细胞。比例尺，50μm。所有结果代表至少三个独立实验中产生的数据。
图6A-6D为PMSC表达高水平的IL-6以保护胰岛β细胞。(6A)PMSCs分泌的IL-6水平明显高于BMMSCs。来自小鼠PMSC和BMMSC的培养上清液的细胞因子阵列分析。取等体积的PMSC和BMMSC培养上清液用于细胞因子阵列(n＝3)。(6B-6D)ELISA，PCR和Western blot分析证实，PMSCs表达的IL-6水平高于BMMSCs。
图7为代表性的免疫荧光图像显示，肾囊膜下植入IL-6敲除的PMSC无法挽救1型糖尿病小鼠的受损原代胰岛(如胰岛素和Ki67染色)。黑色虚线表示胰岛。右图是标记物 +细胞的定量，n＝5。比例尺，200μm，低倍放大。
图8A-8D为PMSC表达高水平的IL-6以保护β细胞。(8A)流式细胞仪分析表明，大多数CD105 +PMSC表达IL-6。(8B)胰腺组织的免疫染色显示在外分泌和内分泌组织中均检测到IL-6。(8C,8D)PMSC在体外保护MIN6β细胞免受STZ诱导的细胞凋亡。在5-10mg/mL STZ处理24小时后，将MIN6β细胞与PMSC或BMMSC共培养。通过用annexin V和7AAD标记，测量MIN6细胞的凋亡，n＝3。在培养24小时的活(8C)或凋亡(8D)的MIN6β细胞上门控的代表性流式细胞仪图。MIN6组：STZ处理MIN6β细胞24小时后，继续培养；PMSC+MIN6：STZ处理MIN6β细胞24小时后，与PMSC共培养；BMMSC+MIN6：STZ处理MIN6β细胞24小时后，与BMMSC共培养(直接接触，BMMSC对β细胞有杀伤作用)。
图9A-9E为探讨IL-6在STZ诱导的糖尿病小鼠的PMSC介导的治疗中发挥关键作用。(9A,9B)与野生型PMSC植入相比，IL-6基因敲除的PMSC的肾囊膜下植入未能改善1型糖尿病表型。植入后1个月使用腹腔注射葡萄糖耐量实验(IPGTT)检测小鼠葡萄糖清除能力，n＝3。使用尾静脉血液样本随时间测量指定组小鼠的血糖水平。(9C)肾囊膜下植入的IL-6基因敲除的PMSCs局部再生了腺泡结构，可检测到胞内散在分布嗜碱性染色的腺泡样细胞。(9D，9E)组织学和免疫染色分析进一步证实，IL-6基因敲除的PMSC植入不能挽救STZ诱导的糖尿病小鼠胰腺组织中受损的胰岛，胰岛素阳性细胞的数量。
图10为PMSC表达高水平的IL-6以保护β细胞。IL-6敲除的PMSCs在体外保护MIN6β细胞免受STZ诱导的细胞凋亡的能力下降。在5-10mg/ml STZ处理24小时后，将MIN6β细胞与野生型或IL-6敲除PMSCs共培养。通过使用流式细胞术用annexin V和7AAD标记来测量MIN6细胞的凋亡。
图11A-11D为PMSCs的肾囊膜下植入提高了IL-6的水平。(11A)PMSCs的肾囊膜下植入表达高水平的IL-6。代表性的免疫荧光染色显示野生型PMSCs植入组中具有强烈的IL-6免疫染色。在IL-6敲除的PMSCs植入组中没有观察到IL-6染色。(11B)ELISA分析显示，PMSCs植入后2周和4周血清IL-6水平升高。虚线表示对照组小鼠的基础血清IL-6水平。(11C)植入IL-6敲除的PMSCs未能抑制IL-17的水平。其中，在植入后4周，采用ELISA方法对原位胰腺中的IL-10，IL-17，TGF-β和IL-6进行分析。(11D)植入IL-6敲除的PMSCs未能抑制1型糖尿病小鼠的系统中Th17细胞的激活。通过流式细胞仪分析显示1型糖尿病小鼠脾脏CD4 +IL-17 +Th17细胞和CD4 +T细胞中的CD25 +Foxp3 +Treg细胞。所有结果代表至少三个独立实验中产生的数据。
图12A-12D为IL-6敲除的小鼠表现出对PMSCs植入疗法的抵抗力。(12A-12B)IL-6敲除小鼠STZ诱导的1型糖尿病的严重程度增加，并对PMSC植入疗法产生抗药性。(12C，12D)1型糖尿病IL-6敲除小鼠接受PMSCs的肾囊膜下植入后的宿主原位胰腺/胰岛的代表性H&E图像。所有结果均代表在至少三个独立实验中产生的数据。误差线＝平均值±SD。使用单向方差分析和Bonferroni校正或独立的非配对两尾学生t检验分析数据。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
图13(13A)与单独肾囊膜下植入BMMSC组相比，BMMSC肾囊膜下植入治疗的同时腹腔注射细胞因子IL-6组小鼠血糖降低的更加显著。用多次低剂量链脲佐菌素(STZ，五剂50mg/kg体重)注射液诱导野生型小鼠糖尿病。BMMSC+IL-6组小鼠除了肾囊膜下植入BMMSC以外，在移植MSC当天以及随后每周，连续四次给予腹腔注射IL-6(2ng/ml,每只小鼠每次注射200μl)。在如图所示时间点，使用尾静脉血液样本测量指定组小鼠的血糖水平，n＝4。(13B)与单独肾囊膜下植入BMMSC组相比，BMMSC肾囊膜下植入治疗的同时腹腔注射细胞因子IL-6组小鼠葡萄糖清除能力更强。植入后1个月使用腹腔注射葡萄糖耐量实验(IPGTT)检测小鼠葡萄糖清除能力，n＝3。
具体实施方式
以下通过具体的实施例进一步说明本公开的技术方案，具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
“土壤挽救种子”策略：尽管胰腺外分泌腺来源的间充质干细胞(PMSC)表达了多种胰腺祖细胞标记，包括PDX1，Nkx6.1和Ngn3，但当 植入肾囊时，它们主要再生胰腺外分泌腺(土壤)，再生组织中仅有极少量的胰岛素分泌细胞(种子)。这些异位PMSC植入物不产生胰岛素，但能够通过拯救受体受损的β细胞来改善1型糖尿病表型。从机理上讲，我们发现PMSCs通过表达高水平的IL-6来抑制IL-17，从而改善胰腺免疫微环境(土壤)，进而挽救受损的胰岛β细胞(种子)。此外，我们发现与静脉内注射PMSCs相比，PMSCs的肾囊植入在STZ诱导的小鼠中显示出显著且持续的治疗效果。PMSC介导的外分泌胰腺再生可挽救1型糖尿病小鼠胰岛β细胞的受损。这项研究揭示了外分泌胰腺再生在保护β细胞方面的重要作用，并提供了1型糖尿病治疗的新的“土壤拯救种子”的策略。
“STZ”表示链脲佐菌素(streptozotocin)。
所述“胰蛋白酶”和“胰脂肪酶”为胰腺外分泌腺特异性标志物，所述“胰岛素”为胰岛β细胞标志物。
所述腺泡和导管细胞标志物如淀粉酶、胰蛋白酶、胰脂酶和CK19等。
如本文所使用的术语“和/或”是指并且涵盖一个或多个相关联的所列项目的任何和所有可能的组合。当在两个或多个项目的列表中使用时，术语“和/或”表示所列出的项目中的任何一个可以单独使用，或者可以使用两个或多个所列出的项目的任何组合。例如，如果组合物，组合，构造等被描述为包括(或包含)组分A，B，C和/或D，则该组合物可以单独包含A；单独包含B；单独包含C；单独包含D；包含A和B的组合；包含A和C的组合；包含A和D的组合；包含B和C的组合；包含B和D的组合；包含C和D的组合；包含A，B和C的组合；包含A，B和D组合；包含A，C和D的组合；包含B，C和D组合；或A，B，C和D组合使用。
在本公开中，“组合物”中的组分可以是混合的形式存在，也可以被分开包装。分开的包装的组分也可以含有其各自的佐剂。所述的佐剂是指在药学中，可辅助药物疗效的手段。对于组合物中的组分在分开包装的情况下，各个分开包装的组分可以是同时施用或者是以任意的前后顺序施用，其中患者先用一种药物治疗，然后再给以另一种药物。所述的患者是指哺乳动物受治疗者，尤其是人类。
如本文所用，除非另有说明，术语“治疗”，是指当患者患有特定疾病时发生的作用，疾病或病症，其降低了疾病，病症或病症的严重程度，或延缓或减缓了疾病，病症或病症的发展(“治疗性治疗”)，并且还考虑了在患者开始患有特定的疾病，病症或病症之前发生的动作(“预防性治疗”)。
以下对应实验结果图的实验方法如未特别说明都是按照商品化试剂盒或现有技术的实验标准流程进行，相关的实验方法举例如下所述，以下实施例中涉及到的实验技术方法如未特别说明，都是通用的。
实验动物：雌性C57BL/6J，NU/j，NOD/SCID，IL-6基因敲除，小鼠用于本实验。在所有实验中均使用具有相同背景的年龄匹配的8至10周龄的雌性小鼠。所有动物实验均根据机构批准的用于动物研究的方案进行(中山大学SYSU-IACUC#2020000122和宾夕法尼亚大学IACUC#805478)。在MSC植入后按照各实验所显示的时间对小鼠实施安乐死。为了破坏内源性胰岛细胞诱导1型糖尿病，使用200mg/kg体重的单次高剂量或50mg/kg连续五天底剂量的腹膜内(ip)注射链脲佐菌素(STZ)，用0.01M柠檬酸钠在pH 4.5下溶解STZ，注射于8周大小鼠。使用Optium Xceed血糖仪，取小鼠尾静脉血液样本测量循环葡萄糖水平。连续两次测量的非空腹血糖≥300mg/dl被认为表明是显著的糖尿病特征(77)。为了进行腹膜内葡萄糖耐量试验(IPGTT)，将动物禁食5小时，然后腹膜内注射。注射20％葡萄糖溶液(每公斤体重2克葡萄糖)。注射后0、30、60、90和120分钟测量血糖。
流式细胞术：
对于CD4、Foxp3染色，收获脾细胞并在冰上用抗CD4-PerCP和CD25-APC抗体染色30分钟。接下来，使用Foxp3染色缓冲液试剂盒(eBioscience，圣地亚哥，加利福尼亚，美国)用抗Foxp3-PE抗体对细胞染色，以进行细胞固定和通透化。对于IL-17A等细胞因子染色，用500ng/ml PMA和500ng/ml离子霉素(Peprotech，Rocky Hill，NJ，美国)处理脾细胞5小时，并加入布雷菲德菌素A(BFA；5μg/ml，Peprotech)，在培养的最后4小时内。随后，将细胞用抗CD4-PerCP抗体染色，然后使用染色缓冲液试剂盒用抗IL17A PE抗体染色。所有抗体均购自BioLegend(美国加利福尼亚州圣地亚哥)。用FACScalibur流式细胞仪(BD Bioscience，San Jose，CA，USA)分析样品。
组织学：
将解剖的胰腺和肾脏样品固定在4％多聚甲醛(Sigma-Aldrich)中，然后石蜡包埋。石蜡切片(5μm)用苏木精和曙红(H&E)染色。为了进行免疫组织化学染色，将石蜡包埋的切片用5％BSA封闭，与针对IL-6的一抗在4℃下孵育过夜，然后使用VECTASTAIN UNIVERSAL Elite ABC试剂盒以及ImmPACT VIP过氧化物酶底物试剂盒(VECTOR)，按照制造商的说明进行染色。
免疫荧光：
为了制备冷冻切片样品，将切开的胰腺在4℃的4％PFA中固定4个小时，用PBS洗涤3次，然后包埋在最佳切割温度(OCT)化合物中。将组织切片与一抗在4℃孵育过夜，然后在PBST(PBS+0.1％Triton X-100)中洗涤，再与二抗和DAPI在室温下孵育2小时，然后洗涤并固定以进行共聚焦成像。为了进行全壁染色，将胰岛在4℃的4％PFA中固定1小时，用PBS洗涤3次，然后在室温下用全壁封闭缓冲液封闭1小时。封闭缓冲液在PBST中含有10％FBS。将一抗稀释于封闭缓冲液中，并在4℃下孵育过夜，然后用PBST洗涤3次，然后将二抗和DAPI在4℃下孵育过夜。使用激光共聚焦显微镜拍摄照片。
细胞因子阵列分析和酶联免疫吸附测定(ELISA)：
根据制造商的说明，使用购自R&D Systems(美国明尼苏达州明尼阿波利斯)的ELISA试剂盒测量了小鼠的血清及组织样品中的各细胞因子及C-peptide水平。
细胞因子阵列分析。根据制造商的说明，使用Mouse Cytokine Array Panel A Array Kit(R&D Systems，Minneapolis，MN，USA)，取等体积的BMMSC和PMSC进行分析。使用Image J软件对结果进行扫描和分析，以计算印迹强度。
蛋白质印迹(Western blotting):
将细胞在含有蛋白酶和磷酸酶抑制剂(Roche，巴塞尔，瑞士)蛋白提取试剂(Thermo，Waltham，MA，美国)中裂解，并使用蛋白浓度测定法(Bio-Rad Laboratories，Hercules，美国加利福尼亚州)测定蛋白浓度。使用SDS-PAGE胶垂直电泳分离20μg蛋白质，并转印到0.2μm硝酸纤维素膜上(Millipore，Burlington，MA，美国)。将膜用5％脱脂奶粉和0.1％Tween-20封闭1小时，然后根据制造商的说明，将第一抗体在封闭溶液中稀释过夜。然后将膜在封闭液中以1：10,000稀释的HRP偶联二抗(Santa Cruz)中于室温孵育1小时。使用SuperSignal West Pico化学发光底物(Thermo)和Bio-Rad ChemiDoc TM成像仪(Hercules，CA，美国)检测蛋白表达。
实时定量PCR(Real-Time PCR.)：
使用TRIzol试剂盒(Life Technologies，Invitrogen)从不同的细胞中分离提取总RNA。RNA样品(1μg)在逆转录系统(QIAGEN)中逆转录。PCR实验所使用的引物为：小鼠IL-6上游引物GGCGGATCGGATGTTGTGAT，下游引物GGACCCCAGACAATCGGTTG。PCR条件为：95℃ 5分钟，(95℃ 10秒，50℃ 45秒)×40和90℃ 10秒。
体外CD4+IL-17A+Th17细胞诱导：
根据制造商的说明，使用BD IMag TM抗小鼠CD4Particles-DM(BD Bioscience)从脾细胞中分离出CD4+T淋巴细胞。用结合抗CD3e抗体的培养板(克隆145-2C11，5μg/ml，BioLegend)和可溶性抗CD28抗体(克隆37.51，5μg/ml，BioLegend)预刺激CD4+T细胞2天。对于Th17细胞(T辅助细胞17)的诱导，将活化的T细胞(1×106)加入重组人TGF-β1(2μng/ml；R&D Systems，明尼阿波利斯，明尼苏达州，美国)的0.2×106MSCs小鼠IL-6(25μng/ml；Peprotech)，重组小鼠IL-23(20μng/ml；Peprotech)和重组小鼠IL-β1(20ng/ml；Peprotech)(89)。3天后，收集悬浮液中的细胞用于IL-17A抗体染色，并用FACScalibur(BD Bioscience)分析。
实施例1 胰腺间充质干细胞(PMSCs)的分离培养与鉴定
分离培养的步骤包括：
将小鼠的胰腺组织轻轻地分离，切碎，并用含有2mg/mL I型胶原酶(Worthington Biochemical，Lakewood，NJ，美国)和4mg/mL Dispase II(Roche Diagnostics，Basel，瑞士)的磷酸盐缓冲液(PBS)在37℃放置1小时消化。使消化所得细胞悬液通过70μm滤器(BD Biosciences，美国加利福尼亚州圣何塞)获得胰腺的单细胞悬液。将所有核细胞(ANC)接种到100mm培养皿中，该完全培养基含有α-MEM(Invitrogen)，并添加了20％FBS，2mM L-谷氨酰胺(Invitrogen)，55μM 2-巯基乙醇(Invitrogen)，100U mL -1青霉素和100μgmL -1链霉素(Invitrogen)，然后在37℃和5％CO 2下孵育48小时。用PBS洗涤培养物两次以去除非贴壁细胞。将贴壁的细胞在相同条件下在上述完全培养基中再培养12天。
进一步对PMSCs进行鉴定，并证明PMSCs具有自我更新的能力(通过CFU-F评估)和高增殖率(通过Brdu染色评估)：
(1)流式细胞仪分析证实这些胰腺组织来源的CD105 +PMSCs共表达β细胞标志物胰岛素(insulin)、腺泡细胞标记淀粉酶(acinar cell marker amylase)、导管细胞标记物(pancreatic progenitor cell markers)CK19、胰腺祖细胞标志物(pancreatic progenitor cell markers)Ngn 3、Ptf1a、Hnf1β、PDX1和Nkx6.1(图1A)。另外，流式细胞仪分析证实这些胰腺组织来源MSCs阳性表达间充质干细胞表面标记物CD44，CD105，CD73和Sca-1，但不表达血液细胞标记物CD34和CD45(图1B)。
(2)这些MSCs还显示出了多能分化的能力，包括成骨，成脂(图1C，1D)，成骨诱导及染色步骤如下：
1)用常规培养基培养直至细胞融合；2)每周2-3次更换成骨培养基，持续4周或更长时间；3)当观察到矿化的结节时，用茜素红S染色；4)蛋白质收集时间为诱导后1周；5)将1g茜素红S溶于100ml蒸馏水中；6)通过0.22um过滤器过滤并在室温下储存；用PBS清洗培养皿2次；7)在室温下用60％异丙醇固定1分钟；8)用蒸馏水补水2-3分钟；9)在室温下用染色溶液染色3分钟；10)用蒸馏水洗培养皿数次；11)干燥，并在显微镜下观察，使用Image J软件计算成骨阳性区域。
其中，成脂诱导和染色同样按照商品化试剂盒的实验标准流程进行。(3)通过CFU-F评估的自我更新能力(图1E左侧)和使用Brdu染色评估细胞增殖率(图1E右侧)。其中，CFU-F评估的实验方法如下：1)将60毫米培养板接种从胰腺中分离出的5×10 3个单个核细胞(ANC)；
2)14天后，将培养板用0.1％甲苯胺蓝和2％多聚甲醛溶液的混合物染色；
3)将含有＞50个细胞的克隆计数为单个克隆簇，进行CFU-F计数。
BrdU标记法的步骤如下：1)细胞以1×10 5/mL细胞数接种于12孔板中(内放置一盖玻片)，培养1天，用含0.4％FBS培养液同步化3天，使绝大多数细胞处于G0期；
2)终止细胞培养前，加入BrdU(终浓度为30μg/L)，37℃，孵育40min；
3)弃培养液，玻片用PBS洗涤3次；
4)甲醇/醋酸固定10min；
5)经固定的玻片空气干燥，0.3％H 2O 2-甲醇30min灭活内源性氧化酶。
5％BSA封闭；
6)变性液5min变性核酸；
7)冰浴冷却后PBS洗涤，加一抗即抗小鼠BrdU单抗；
8)荧光二抗染色，在显微镜下随机计数5个高倍视野中细胞总数及BrdU阳性细胞数，计算阳性率。
从检测结果可以看出，相对于BMMSC而言，PMSC具有更强的CFU-F的形成和增殖能力，更强的成骨分化能力以及较低的成脂分化能力。与BMMSC相似，PMSC表达MSC阳性表面标记物Sca-1，CD105，CD44，CD73不表达血液细胞表面标记物CD34和CD45。
免疫荧光染色显示胰腺外分泌腺来源的MSC(PMSCs)表达成熟的胰岛特异性标记，包括胰岛素(insulin)，以及成熟的外分泌腺标记物淀粉酶(amylase)和导管细胞标记细胞角蛋白19(CK19)(图1F)。此外，通过流式细胞术分析(图1A、1G)、免疫荧光染色(S1B)，PMSCs表达了胰腺祖细胞标记物PDX1，Nkx6.1，以及内分泌分化标记物Ngn 3，腺泡分化标记物Ptf1a和导管分化标记物Hnf1β。有趣的是，流式细胞术检测显示，高达71.11％的CD105 +PMSC表达外分泌标记淀粉酶，而只有11.52％的CD105 +PMSC表达胰岛素(图1A)。这些数据显示PMSC具有较低的胰岛素阳性细胞比例和较高的淀粉酶阳性细胞比例，提示PMSCs同时表达成熟的胰腺组织以及分化的细胞和祖细胞的标志物，但是它们表现出外分泌显性的表型。
PMSCs具有一般的MSC特性，包括MSC表面分子的表达以及自我更新和多能分化的能力。我们检测结果显示，与骨髓MSC(BMMSC)相比，PMSC具有克隆形成(CFU-F)能力，并具有较高的增殖率(图1E)。流式细胞仪分析证实，PMSC阳性表达间充质干细胞表面标记物CD44，CD73，CD105和Sca-1，阴性表达血液细胞标记物CD34和CD45(图1B)。PMSCs还显示出多能分化的能力，包括成骨，成脂分化(图1C、1D)。
实施例2 胰腺间充质干细胞肾囊膜下植入小鼠体内
于10cm细胞培养皿中培养、扩增胰腺间充质干细胞，常规换液至细胞接触融合，弃掉培养液，消化成单个细胞，收集约4×10 6个间充质细胞；用约20μL 1×PBS溶液混悬细胞，将其置于冰上待用；或将上述PBS细胞混悬液与小块可吸收明胶海绵混合，将混合物将置于冰上待用。也可选择于10cm细胞培养皿中培养、扩增胰腺间充质干细胞，常规换液至细胞接触融合后继续培养细胞约3-7天周左右，使其细胞基质变厚，用钝tip头或其他方式从培养皿中整体剥离贴壁的PMSC，将其转移至新的24孔板继续培养，1-3天后形成PMSC小球，将PMSC小球置于冰上待用。(几种方式植入肾囊膜下后再生结果并无明显差异。)
将小鼠麻醉后，背部皮肤去毛、消毒备皮。手术剪在皮肤上形成2厘米左右的纵向切口，暴露肾脏。用显微手术剪或显微镊的尖端于肾囊膜下开口，并钝性分离肾囊膜与膜下肾组织，为植入物制作一个口袋。将PMSC+明胶海绵混合物或PMSC小球置于肾囊膜下袋中，或将PMSC细胞混悬液注射入肾囊膜下。使用电刀封闭肾囊膜创口，将肾脏放回原位，缝合手术创口。8周后收集移植物进行相应检测。
实施例3 胰腺干细胞异位再生外分泌胰腺
当将PMSCs植入野生型或免疫缺陷小鼠的肾囊膜下时，其在植入后8周即可异位再生无内分泌胰岛的胰腺外分泌腺组织(图2A-D)。与正常的胰腺外分泌腺相似，再生的外分泌胰腺由典型的腺泡和导管组成(图2C-E，)。再生的腺泡显示出外分泌胰腺细胞具有强嗜碱性染色特性，并带有嗜酸性的分泌颗粒，并且导管有上皮样衬里(图2C-E)。免疫荧光染色证实，再生的异位胰腺表达外分泌标志物淀粉酶，胰蛋白酶和胰脂肪酶，以及导管标志物CK19(图2E)。大约80％比例的PMSC植入物表现出外分泌胰腺结构，只有12.5％比例的PMSC植入物含有少量散布的产生胰岛素的β细胞(图2A-2F)。在植入物中未检测到其他种类的内分泌细胞，例如产生胰高血糖素的α细胞，产生生长抑素的δ细胞和产生胰多肽的PP细胞(图2A、2E)。这些数据表明大多数PMSC再生植入物形成外分泌胰腺，而没有大量胰岛素产生。
实施例4 PMSC介导的肾囊膜下胰腺再生改善了STZ诱导的I型糖尿病
为了检测PMSC再生的异位外分泌胰腺对糖尿病的治疗效果，我们首先使用多次低剂量链脲佐菌素(STZ)注射液在野生型小鼠中诱导了1型糖尿病。我们比较了PMSC和BMMSC植入肾囊膜下的效果，并发现PMSC植入而非BMMSC植入可显著降低1型糖尿病小鼠的血糖水平(图3A)。此外，与BMMSC组相比，PMSC植入挽救了葡萄糖的清除能力，并提高了血清C肽和胰岛素的水平(图3B-D)。在STZ诱导的糖尿病小鼠肾囊膜下PMSC植入组可检测到再生的外分泌腺泡细胞和腺泡(图3E)。免疫荧光染色证实，PMSC再生的异位胰腺细胞表达外分泌标记淀粉酶(图3E)。为了进一步确认PMSC植入的治疗潜力，我们使用单次大剂量STZ注射诱导了免疫缺陷小鼠的1型糖尿病。与上述结果一致，我们发现植入PMSCs而非BMMSCs可以显著提高1型糖尿病小鼠的存活率(图3F)。PMSC植入后，糖尿病小鼠的寿命从40天延长到120天(图3F，图3G)。PMSC组在植入后60天血糖水平显著降低，而BMMSC组则没有降低(图3G，图3H)。此外，与BMMSC组相比，PMSC植入挽救了葡萄糖清除能力(图3H)。这些数据表明PMSC植入能够拯救STZ诱导的1型糖尿病表型，这可能不依赖于胰岛素的产生。
为了进一步证实PMSC介导的外分泌胰腺再生可改善糖尿病的表型，我们比较了PMSCs肾囊膜下植入与系统回输(小鼠尾静脉注射)PMSC的功效。在PMSC植入组中观察到了肾囊膜下再生的异位外分泌胰腺，而在全身输注组中则未观察到(图3I)。PMSC给药后7天，PMSC肾囊膜下植入组和系统回输组的血糖水平均降低(图3J)。但是，与接受系统回输的PMSC的组相比，PMSC肾囊膜下植入组在治疗后一个月显示出明显的葡萄糖清除能力恢复和血清C肽水平升高(图3K，L)。有趣的是，PMSC给药后40天，肾囊膜下植入组小鼠保持了血糖改善的效果，但系统回输组未能维持血糖水平的降低(图3M)。我们的数据表明，与静脉输注相比，PMSC肾囊膜下植入介导的异位外分泌胰腺再生在STZ诱导的1型糖尿病小鼠中具有显著持续的治疗作用。
实施例5 PMSCs的肾囊膜下植入可挽救1型糖尿病小鼠受损的原位胰腺/胰岛
为了探索PMSC介导的异位外分泌胰腺再生如何拯救糖尿病表型，我们检测了STZ诱导的糖尿病小鼠的原位胰腺。我们发现，通过H&E 染色评估，肾囊膜下PMSC植入显著改善了小鼠原位胰腺组织中胰岛的组织学结构(图4A)。免疫染色显示，胰岛素阳性β细胞以及Ki67和胰岛素双重阳性增殖β细胞的数量均显著增加(图4B，4C)。同样，淀粉酶，胰蛋白酶和胰脂肪酶阳性腺泡细胞也显著增加(图4B，图5A)。从实验结果中看出，是PMSC而非BMMSC的植入挽救了STZ诱导的胰岛β细胞和腺泡细胞的损伤(图4A，4B，4C)。PMSCs经过系统注射，未能显著改善胰岛组织中的胰岛大小，胰岛素阳性细胞数量以及Ki67/胰岛素双阳性增殖β细胞(图4D-F，图5B)。这些数据表明，PMSCs的肾囊膜下植入可以挽救STZ诱导的原发胰腺/胰岛组织损伤，并证明“土壤挽救种子”策略用于1型糖尿病治疗的可行性。
实施例6 PMSC表达升高水平的IL-6以保护β细胞
通过细胞因子阵列分析，ELISA和PCR，Western印迹检测(图6A-D)。我们发现与BMMSCs相比，PMSC显著表达IL-6(一种能够充当促炎或抗炎细胞因子的白介素)(Scheller,J.,Chalaris,A.,Schmidt-Arras,D.&Rose-John,S.The pro-and anti-inflammatory properties of the cytokine interleukin-6.Biochim Biophys Acta 1813,878-888,doi:10.1016/j.bbamcr.2011.01.034(2011).)。免疫染色和流式细胞仪分析证实，大多数CD105阳性的PMSC表达IL-6(图7，图8A)。免疫染色分析显示IL-6在胰腺胰岛和外分泌胰腺中均有表达(图8B)。此外，我们发现PMSCs，而不是BMMSCs，可以在体外共培养系统中保护胰腺β细胞系MIN6免受STZ诱导的细胞凋亡(图8C，8D)。
我们将IL-6敲除小鼠来源的PMSC用于肾囊膜下植入治疗，发现与野生型PMSC植入组相比，IL-6敲除小鼠来源的PMSC未能改善STZ诱导的糖尿病表型(图9A-9C)。肾囊膜下植入IL-6基因敲除的PMSCs部分地再生了腺泡结构，可检测到嗜碱性染色的腺泡样细胞，但是其组织学结构与正常腺泡有差别，且荧光染色显示淀粉酶染色散在，不似PMSC再生组织那么均一(图9C)。组织学和免疫染色分析进一步证实，在STZ诱导的糖尿病小鼠中，IL-6基因敲除的PMSC植入未能挽救受损的胰岛，胰岛素阳性细胞和胰腺组织(图9D-9E)。另外，IL-6基因敲除的PMSCs在体外用STZ处理后，抗MIN6β细胞凋亡的能力显著降低(图10)。
实施例7 PMSCs的肾囊膜下植入提高IL-6的水平
移植后2周，在STZ诱导的糖尿病小鼠肾囊膜下移植物中，在野生型PMSC移植组观察到强烈的IL-6染色，但在IL-6基因敲除PMSC移植组中未观察到IL-6表达(图11A)。有趣的是，如通过ELISA所示，在PMSC植入后2和4周时，肾囊膜下植入野生型而非IL-6基因敲除PMSC升高了血清和原位胰腺中IL-6的水平(图11B)，表明异位植入肾囊膜下的PMSCs上调了胰腺和血清IL-6的水平。接下来，我们发现野生型而非IL-6基因敲除的PMSC植入抑制IL-17的水平(图11C)。相应地，IL-6基因敲除的PMSC植入未能挽救STZ诱导的糖尿病小鼠Th17/Treg细胞水平的改变(图11D)。这些数据表明，IL-6是通过抑制IL-17来改善胰腺免疫微环境”这一通路所必需的。
实施例8 IL-6基因敲除小鼠显示出对PMSC植入疗法的抗性
我们发现IL-6基因敲除小鼠在STZ诱导下显示出严重的糖尿病表型，而异位PMSC移植未能挽救IL-基因敲除小鼠的糖尿病表型(图12A-D)。H&E染色显示，来自IL-6敲除小鼠的胰腺组织显示出与野生型小鼠相似的结构，但肾囊PMSC植入未能挽救STZ诱导的敲除小鼠胰岛β细胞的破坏。这些发现证实了IL-6在保护β细胞中的作用。
实施例9 IL-6在骨髓间充质干细胞肾囊膜下移植治疗1型糖尿病中的作用
用多次低剂量链脲佐菌素(STZ，五剂50mg/kg体重)注射液诱导野生型小鼠糖尿病。骨髓间充质干细胞(BMMSC)+IL-6组小鼠除了肾囊膜下植入骨髓间充质干细胞(BMMSC)(4×10 6细胞每只小鼠)以外，在移植BMMSC当天以及随后每周，连续四次给予腹腔注射IL-6(2ng/mL，每只小鼠每次注射200μL)。在如图13A所示时间点，使用尾静脉血液样本测量指定组小鼠的血糖水平，n＝4。在植入BMMSC后1个月使用腹腔注射葡萄糖，进行糖耐量实验(IPGTT)检测小鼠葡萄糖清除能力，n＝3。
检测结果如图13A，13B)所示，我们在使用骨髓间充质干细胞(BMMSC)肾囊膜下植入治疗的同时腹腔注射细胞因子IL-6。发现与单独肾囊膜下植入BMMSC组相比，BMMSC肾囊膜下植入治疗的同时腹腔注射细胞因子IL-6治疗STZ诱导的糖尿病的效果更佳(图13A，13B)。

Claims (17)

  1. 来源于胰腺外分泌腺的间充质干细胞或胰腺外分泌腺组织在制备治疗和/或预防糖尿病药物当中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述的胰腺外分泌腺组织由胰腺外分泌腺的间充质干细胞生成；
    优选地，所述的药物为注射制剂；
    更优选地，所述药物为肾囊膜下注射制剂。
  3. 如权利要求1-2任一所述的应用，其特征在于，所述糖尿病为Ⅰ型糖尿病。
  4. 一种用于治疗治疗和/或预防糖尿病的系统，其特征在于，包含胰腺外分泌腺的间充质干细胞，以及植入装置；优选地，所述的植入装置用于将胰腺外分泌腺的间充质干细胞植入患者体内定点部位；
    优选地，所述的介入装置为穿刺器；
    优选地，所述的定点部位包括肾囊膜下、皮下、或肠系膜；
    优选地，所述的系统还包含超声装置或内窥镜装置；
    所述超声装置或内窥镜装置用于引导所述穿刺器的定位。
  5. 一种获得胰腺外分泌腺组织的方法，其特征在于，所述的胰腺外分腺的组织通过植入间充质干细胞而形成；
    或优选地，所述的植入包括肾囊膜下植入、皮下植入、肠系膜植入；
    更优选地，所述的植入为肾囊膜下植入；
    优选地，所述的植入为异位植入；
    优选地，所述的异位植入优选为非活体的组织模型植入。
    优选地，所述的间充质干细胞包括胰腺间充质干细胞、骨髓间充质干细胞；
    优选地，所述的间充质干细胞为胰腺间充质干细胞；
    或优选地，所述的间充质干细胞为胰腺外分泌腺的间充质干细胞；
    或优选地，所述的间充质干细胞为游离的间充质干细胞；或优选地，所述的间充质干细胞无需处理直接植入。
  6. 如权利要求5所述的方法，其特征在于，包括以下步骤：
    (1)用培养液培养间充质干细胞；
    (2)去掉步骤(1)中的间充质干细胞的培养液，收集间充质干细胞；
    (3)将步骤(2)中收集到的间充质干细胞用PBS或生理盐水混悬后，植入动物肾囊膜内或皮下，从而获得胰腺外分泌组织；
    优选地，步骤(1)中的培养液的成分包括FBS，谷氨酰胺，2-巯基乙醇和α-MEM；
    更为优选地，步骤(1)中，所述间充质干细胞培养至P1-P3代。
  7. 一种胰腺外分泌腺的组织，其特征在于，所述的胰腺外分泌腺的组织通过权利要求5-6任一所述的方法获得。
  8. 权利要求7所述的胰腺外分泌腺的组织在胰腺相关疾病的治疗药物的发现筛选、或胰腺相关疾病治疗药物的毒性测定、或胰腺胚胎学、或胰腺细胞谱系和分化途径的研究、或基因表达研究、或参与胰腺损伤和修复的机制研究、或胰腺的炎症性疾病研究、或治病机制研究中的体外用途。
  9. 权利要求7所述的胰腺外分泌腺的组织在重组基因表达中的应用。
  10. 含有IL-6和间充质干细胞的组合物在制备治疗和/或预防糖尿病药物方面的应用。
  11. 如权利要求10所述的应用，其特征在于，所述间充质干细胞的来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱、外周血或胰腺；
    优选地，所述间充质干细胞的来源为骨髓；
    或优选地，所述骨髓间充质干细胞为游离的骨髓间充质干细胞。
  12. 如权利要求10-11任一所述的应用，其特征在于，所述IL-6和间充质干细胞被同时给药。
    优选地，所述IL-6和间充质干细胞被单独给药；
    优选地，先给予IL-6，后给予间充质干细胞；
    优选地，先给予间充质干细胞，后给予IL-6；
    优选地，所述药物为注射制剂；
    优选地，所述药物选自静脉注射剂、肌肉注射剂、皮下注射剂或鞘内注射剂；
    优选地，所述间充质干细胞为肾囊膜下注射剂；
    优选地，所述IL-6为腹腔注射剂。
  13. 如权利要求10-12任一所述的应用，其特征在于，所述药物还包括药学上可接受的药用载体；
    优选地，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
  14. 如权利要求10-13任一所述的应用，其特征在于，所述糖尿病为Ⅰ型糖尿病。
  15. 一种用于治疗和/或预防糖尿病的系统，其特征在于，包含间充质干细胞和IL-6，以及植入装置；
    优选地，所述的植入装置用于将间充质干细胞植入患者体内定点部位。
  16. 如权利要求15所述的系统，其特征在于，所述的植入装置为穿刺器；
    优选地，所述定点部位包括肾囊膜下、皮下、或肠系膜；
    优选地，所述的系统还包含超声装置或内窥镜装置；
    优选地，所述超声装置或内窥镜装置用于引导所述植入装置的定位。
  17. 一种治疗糖尿病的方法，其特征在于，包括给予患者来源于胰腺外分泌腺的间充质干细胞或移植权利要求7所述的胰腺外分泌腺组织或含有IL-6和间充质干细胞的组合物；
    优选地，所述糖尿病为Ⅰ型糖尿病；
    优选地，所述的胰腺外分泌腺的间充质干细胞通过肾囊膜下植入、皮下植入、肠系膜植入给予所述的患者；
    优选地，所述植入为异位植入；
    优选地，所述异位植入为非活体的组织模型植入；
    优选地，所述IL-6和间充质干细胞的组合物中，所述间充质干细胞的来源包括骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱、外周血或胰腺；
    优选地，所述骨髓间充质干细胞为游离的骨髓间充质干细胞；
    优选地，所述IL-6和间充质干细胞被同时给药。
    优选地，所述IL-6和间充质干细胞被单独给药；
    优选地，先给予IL-6，后给予间充质干细胞；
    优选地，先给予间充质干细胞，后给予IL-6；
    优选地，所述组合物为注射制剂；
    优选地，所述组合物选自静脉注射剂、肌肉注射剂、皮下注射剂或鞘内注射剂；
    优选地，所述组合物中，所述间充质干细胞为肾囊膜下注射剂；
    优选地，所述组合物中，所述IL-6为腹腔注射剂。
    优选地，所述组合物还包括药学上可接受的药用载体；
    优选地，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
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