Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell

A stem cell line and islet cell technology, applied in the field of cell engineering, can solve the problems of ineffective treatment of diabetes and low differentiation efficiency, and achieve the effects of not being prone to aging and apoptosis, low nutritional requirements, and maintaining division and proliferation

Inactive Publication Date: 2012-05-02
厚朴生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 pancreatic stem cell line from human insulin and differentiating to insulin secretion cell
  • Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell
  • Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0052] 1) Aseptically collect the aborted fetal pancreas, place it in pre-cooled Hank's, rinse and remove the peripheral blood and cut off the surrounding fat, blood vessels, tissue capsule and connective tissue, then cut into tissue pieces of about 1mm3, add 0.1 %Collagenase type IV, digested at 37°C for 20-30min. Oscillate from time to time, pass through 80-mesh sieve, centrifuge (500r / min) for 3min, and wash twice, add 20% FBS, 1mM sodium pyruvate, 10mM nicotinamide, 0.1 The RPMI1640 medium of mM β-mercaptoethanol, 2mM glutamine, 20ng / mLEGF, 20ng / mL bFGF and 100mg / mL penicillin / streptomycin was cultured for 48h.

[0053] 2) Manually pick out islet cell clusters under a microscope, further digest them with 0.25% trypsin to form a single-cell suspension, stop d...

Embodiment 2

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

[0056] 2.1 Morphological characteristics of cells

[0057] Morphological changes of pancreatic stem cells derived from human islets were observed under a phase-contrast microscope. Specific results such as figure 1 As shown, among them, panel A is the primary isolated human islet cell group, and panel B is the expanded human islet-derived pancreatic stem cells (pPSCs). Compared with islet cells, the cells are fibroblast-like, mostly triangular and Short fusiform, arranged in a network, and there is contact inhibition between cells. Figure C shows the Giemsa staining of human pancreatic stem cells. The cells are mononuclear, and the nucleus is relatively obvious. There are 2 or more nucleoli, which can be seen Obvious cell division phase. The cells still have good vigor after more than 50 passages in vitro, maintain division and proliferation, and are not prone to senescence and apopto...

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 of self-renewal and multidirectional differentiation. Isolating and cloning pancreatic stem cells in vitro, and inducing their differentiation into functional islets for 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 islets to differentiate them into islet-like cell clusters, which are used to treat nude mouse diabetes models, through related molecular and immunofluorescence detection, high glucose-stimulated insulin release test and transplantation of nude mouse diabetes The model detects its blood sugar level.

[0089] 3.1 Induction, differentiation and identification of neural cells

[0090] Digest the cells with 0.25% trypsin, in...

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Abstract

The invention discloses a method for constructing and differentiating a pancreatic stem cell line from human insulin. The pancreatic stem cell line is a pancreatic stem cell amplified from human insulin mass and has a multi-lineage differentiation potential. The induced pancreatic stem cell line can be differentiated to form nerve-like cells, fat-like cells, bone-like cells and functional insulin-like cell mass. The functional insulin-like cell mass is transplanted to a body of a nude mouse model suffered from diabetes, so that the blood sugar concentration of the nude mouse model can be reduced and normal blood sugar level can be maintained within one month.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a method for constructing a pancreatic stem cell line derived from human islets and differentiating it into insulin-secreting cells. 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 short...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/074C12N5/071C12N5/079C12N5/077A61K35/39A61P3/10
Inventor 曹晖徐军军孙振华
Owner 厚朴生物科技(苏州)有限公司
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