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Method of establishing rat pancreatic duct epithelioid stem cell system

A technology of pancreatic duct and stem cell line, applied in the field of animal cell (tissue) engineering, can solve problems such as immaturity

Inactive Publication Date: 2014-06-25
GUANGDONG OCEAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the establishment of rat pancreatic ductal epithelial-like stem cell lines in China, and the method for establishing rat pancreatic ductal epithelial-like stem cell lines is still immature

Method used

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  • Method of establishing rat pancreatic duct epithelioid stem cell system
  • Method of establishing rat pancreatic duct epithelioid stem cell system
  • Method of establishing rat pancreatic duct epithelioid stem cell system

Examples

Experimental program
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Embodiment 1

[0052] 1. Isolation and culture of primary epithelial-like stem cells from rat pancreatic duct

[0053] The pancreas tissue of adult SD rats was aseptically collected, chopped to 1 mm3, digested with 0.1% type IV collagenase at 37°C for 20 min, and terminated with RPMI1640+10% NBS medium. Filter through a 100-mesh filter, centrifuge (1000rpm, 5min), and wash twice with PBS(-). Resuspend the cells in 25% Dextron separation medium, add 23%, 20%, 11% Dextron separation medium and PBS(-) in sequence, centrifuge with discontinuous density gradient (1500rpm, 5min), collect 11% and 20% Dextron separation The cells were placed in liquid, centrifuged (1000rpm, 5min), and washed. RPMI1640+10%NBS diluted cells to 1×10 6 cells / mL, inoculated on a Petri dish covered with 0.1% gelatin, placed at 37°C, 5% CO 2 , in a saturated humidity incubator. From 3 to 5 days, the primary epithelial-like stem cells of rat pancreatic duct adhered to the wall ( figure 1 ).

[0054] 2. Purification of...

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Abstract

The invention relates to a method of establishing a rat pancreatic duct epithelioid stem cell system, and belongs to the field of animal cell (tissue) engineering. An adult rat pancreatic tissue is digested by collagenase, Dextron discontinuous density gradient centrifugation is carried out, serum culture is carried out in RPMI1640, pancreatic duct primary epithelioid stem cells are grown in a manner of being adhered to the wall, clone ring screening is carried out, epithelioid stem cells are purified, trypsin digestive juice is digested and gone down to the future generation to establish the cell system. The rat pancreatic duct epithelioid stem cell system established by adopting the method disclosed by the invention has the characteristics that the cell adherence is polygonal and epithelioid, and the cells grow in a clonal manner and are normal diploid cells; the rat pancreatic duct epithelioid stem cells express proliferating cell nuclear antigen, an octamer-binding transcription factor 4, stem cell key protein, keratin 19 and a neurogenic differentiation factor 2 on the protein level, have multiple differentiation potentials and are free of oncogenicity.

Description

technical field [0001] The invention relates to a method for establishing human and other mammalian pancreatic ductal stem cell lines, in particular to a method for establishing rat pancreatic ductal epithelial stem cell lines, which belongs to the field of animal cell (tissue) engineering. Background technique [0002] Diabetes mellitus (DM) is one of the major diseases that endanger human health. According to statistics, the number of diabetic patients in the world has reached 346 million (Am J Stem Cells.2012,1(3):196-204.), while the traditional Drugs and insulin replacement therapy can not fundamentally cure the disease. In the past ten years, with the development of transplantation technology, pancreatic islet transplantation has achieved certain curative effect in the treatment of diabetes (N Engl J Med.2000,343(4):230-238. Diabetes. 2005, 54(7): 2060-2069. Am J Transplant. 2008,8(11):2463-2470. Immune Netw. 2013,13(6):235-239.). However, due to the shortage of dono...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 效梅安立龙
Owner GUANGDONG OCEAN UNIVERSITY
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