Method for constructing human islet-derived pancreatic stem cell line and method for differentiating pancreatic stem cell line into pancreas islet cells

Abstract

The invention discloses a method for constructing a human islet-derived pancreatic stem cell line. The method comprises the following steps: S1-1, carrying out resuscitation treatment on human islet-derived cells; and S1-2, adding human pancreatic cells into a culture solution A to be cultured for 4 days, wherein the culture solution A comprises a DMEM culture medium, and the DMEM culture medium contains 15% of FBS, 0.00001 U of penicillin, 0.00001 g of streptomycin, 1 mM of sodium pyruvate, 0.1 mM of non-essential amino acid, 2 mM of glutamine, 0.1 mM of mercaptoethanol and 1000 u / ml of leukemia inhibition factors. A method for differentiating the human islet-derived pancreatic stem cell line into pancreatic islet cells comprises the steps that S4-1, the pancreatic islet stem cell line is subcultured in an H-DMEM / F12 culture medium in the form of suspended cell balls, the H-DMEM / F12 culture medium comprises epidermal growth factors with the concentration being 20 ng / ml, basic fibroblast growth factors with the concentration being 40 ng / ml and 1% of B27, and the concentration of glucose in the culture medium is 17.3 mM. The rejection reaction of the obtained insulin-secreting cells is greatly reduced, and after the cell line is directionally differentiated into the insulin-secreting cells under the culture condition containing LIF, the blood sugar can be effectively reduced.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for constructing a pancreatic stem cell line derived from human islets. Background technique [0002] Diabetes is the third most common chronic disease after cardiovascular disease and cancer, which seriously endangers human life and health. At present, although there are many methods for the treatment of diabetes, such as oral hypoglycemic drugs, injection of exogenous insulin, combined with diet restriction and physical exercise, none of them can fundamentally solve the problem. Organ transplantation is a better solution, but its application is limited by shortage of donors and immune rejection. Stem cells have strong proliferative ability, sufficient quantity, and relatively low transplant rejection. Therefore, it is hoped that stem cell replacement therapy can solve the problems of donor shortage and immune rejection. [0003] Isolating and culturing panc...

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Problems solved by technology

Northwest Agriculture and Forestry University Xiaomei (Establishment of the isolation and culture system of monoclonal human pancreatic stem cells[J]. Molecular Cell Biology, 2008, 41(6):450-456.), Zhao Ting (Human pancreatic stem cells induced islet-like cell clusters and its effect on treating diabetes in rats[J] · Chinese Tissue Engineering Research and Clinical Rehabilitation, 2007, 11(7): 1259-1262 ·), Yao Zhongxiang of the Third Military Medical University (In vitro isolation, culture, identification and differentiation of human pancreatic stem cells Characteristics [J]. Journal of Third Military Medical University, 2003, 25 (I) = 23-25.) Jilin University and others have successively done research on the separation and purification of pancreatic stem cells, the screening of medium, and the identification of cell biology and molecular biology. A lot of work; the isolated pancreatic stem cells formed islet-like cell clusters after directional induction in vivo, and the secretion of insulin and C-peptide was detected, but the differentiation efficiency was very low, and the therapeutic effect on diabetes after transplantation was not significant. Therefore, we propose a method for the construction of human islet-derived pancreatic stem cell lines to solve the above problems