Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells

A stem cell line and islet cell technology, applied in the field of cell engineering, can solve the problems of insignificant treatment effect of diabetes, low differentiation efficiency, etc., and achieve the effects of not easy aging and apoptosis, low nutritional requirements, and improved efficiency.

Active Publication Date: 2013-01-30
苏州厚朴惠康生物医药科技有限公司
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  • Description
  • Claims
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Problems solved by technology

Northwest Agriculture and Forestry University Xiaomei (Establishment of a monoclonal human pancreatic stem cell isolation and culture system [J]. Molecular Cell Biology, 2008, 41(6): 450-456.), Zhao Ting (Human pancreatic stem cell-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 from 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(1): 23-25.) Jilin University and others have successively done research on the isolation 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

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  • Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells
  • Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells
  • Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells

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Experimental program
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Effect test

Embodiment 1

[0050] Example 1. Construction of pancreatic stem cell line derived from human islets

[0051] The construction of a pancreatic stem cell line derived from human islets specifically includes the following steps:

[0052] 1) With the patient’s consent, the excised human pancreatic tissue was collected aseptically during the pancreatectomy of the acute pancreatitis patient who needs partial pancreatectomy, and placed in pre-cooled Hank's to rinse and remove the periphery Cut the blood and the surrounding fat, blood vessels, tissue envelope and connective tissue, and then cut into 1mm 3 Add 0.1% type IV collagenase to the left and right tissue pieces and digest them at 37°C for 20-30 minutes. Vibrate from time to time during the period. After stopping the digestion with 10% FBS Hank's, pass through an 80-mesh screen, centrifuge (500r / min) for 3min, and wash twice, add 20% FBS, 1mM sodium pyruvate, 10mM nicotinamide, 0.1 mM β-mercaptoethanol, 2mM glutamine, 20ng / mL EGF, 20ng / mL bFGF a...

Embodiment 2

[0055] Example 2. Identification of pancreatic stem cell lines derived from human islets

[0056] 2.1 Cell morphology characteristics

[0057] Observe the morphological changes of pancreatic stem cells derived from human islets under a phase contrast microscope. Specific results such as figure 1 As shown, Figure A is a primary isolated human pancreatic islet cell cluster, and Figure B is an expanded human pancreatic islet-derived pancreatic stem cell (hPSC). Compared with pancreatic islet cells, the cells are fibroblast-like, mostly triangular and Short fusiform, arranged in a network, there is contact inhibition between cells. Figure C shows the Giemsa staining of human pancreatic stem cells. The cells are mononuclear and the nuclei are more obvious. There are 2 or more nucleoli, which can be seen Obvious cell division phase. The cell has good viability after passage in vitro for more than 50 generations, still maintains division and proliferation, and is not prone to senescence...

Embodiment 3

[0086] Example 3. Functional characteristics of pancreatic stem cell lines derived from human islets

[0087] Pancreatic stem cells are early developmental cells that exist in pancreatic tissue and have the potential for self-renewal and multidirectional differentiation. Isolating and cloning pancreatic stem cells in vitro and inducing them to differentiate into functional islet transplantation to treat diabetes is an effective way to solve the shortage of islet donors.

[0088] The present invention induces pancreatic stem cell lines derived from human pancreatic islets to differentiate into pancreatic islet-like cell clusters, which are used to treat diabetes in nude mice, through related molecular and immunofluorescence detection, high glucose stimulated insulin release test and transplantation of diabetes in nude mice The model detects its blood sugar level.

[0089] 3.1 Induced differentiation and identification of neural cells

[0090] Digest with 0.25% trypsin, inoculate the c...

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Abstract

The invention discloses a method for constructing a human islet-derived pancreatic stem cell line and a method for differentiation of the human islet-derived pancreatic stem cell line into insulin-producing cells. The pancreatic stem cell line is obtained by amplifying pancreatic stem cells in human pancreatic clusters, and is a potential source for multidirectional differentiation. When induced, the pancreatic stem cell line can differentiate to form neuron-like cells, adipose-like cells, osteoblast-like cells and functional islet-like cell clusters. By transplanting the functional islet-like cell clusters into a body of a diabetic nude mouse model, the blood glucose concentration of the nude mouse model can be reduced and reach a normal level within 24 days.

Description

Technical field [0001] The invention belongs to the technical field of cell engineering, and particularly relates to a method for constructing a pancreatic stem cell line derived from human pancreatic islets and differentiating into insulin secreting cells. Background technique [0002] Diabetes is the third most frequent chronic disease after cardiovascular disease and cancer. It 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 dietary restrictions and physical exercise methods, they cannot fundamentally solve the problem. Organ transplantation is a better solution, but its application is limited by the shortage of donors and immune rejection. Stem cells have strong proliferative power, sufficient numbers, and relatively low transplant rejection. Therefore, people hope that stem cell replacement therapy can solve the problem of don...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071C12N5/079C12N5/077A61K35/39A61P3/10
Inventor 曹晖徐军军孙振华
Owner 苏州厚朴惠康生物医药科技有限公司
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