Mouse islet separation and purification method

A technology for separating and purifying pancreatic islets, which is applied in the field of separating and purifying mouse pancreatic islets. It can solve problems such as harmful effects on cells, reduce time and price, improve the success rate of puncture, and save time.

Active Publication Date: 2016-03-02
SOUTHEAST UNIV
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Problems solved by technology

Moreover, the osmotic pressure of Ficoll is slightly higher than the cell osmotic pressure, which has a certain degree of harmful effects on the cells in the islets.

Method used

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  • Mouse islet separation and purification method
  • Mouse islet separation and purification method
  • Mouse islet separation and purification method

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Experimental program
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Embodiment

[0030] Materials: The experimental animals were dead male ICR mice aged 6-8 weeks, weighing 25-40 g, purchased from the Comparative Medicine Center of Yangzhou University, and fed at the Experimental Animal Center of Southeast University.

[0031] Reagents: RMPI1640 medium (gibco company), fetal bovine serum (gibco company), collagenase Ⅴ (Sigma company), dithizone (Sinopharm Chemical Reagent Co., Ltd.), apoptotic acridine orange staining kit (Kai Bio-based), apoptosis PI staining kit (KGI Bio), penicillin, streptomycin (Hyclone), (LymphotcyteSeparationMedium, American MP company), mouse insulin quantitative analysis enzyme-linked immunoassay kit (Shanghai Qiaoyi Biotechnology Co., Ltd.).

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Abstract

The invention discloses a mouse islet separation and purification method. A self-made puncture needle is adopted to reversely puncture a common bile duct and inject collagenase V to digest a pancreatic gland, and inslets are separated and purified by using the combination of density gradient medium centrifugation and manual sorting. Inset density is identified by dithizone staining, and inset activity is identified by AO/PI staining. Inset functionality is detected by a glucose stimulated insulin release experiment. By using the method to extract islets on a large scale, time can be greatly saved, the puncture success rate can be increased, and Ficoll400 formulation time and price can be reduced. In addition, all the yield, purity, activity and function of islets obtained by purification are relatively good, and the method is simple, fast, cheap and efficient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and purifying mouse pancreatic islets. Background technique [0002] With the development of the economy and the improvement of people's material level, the prevalence of diabetes mellitus (DM) is also increasing. As of 2010, the prevalence of DM in Chinese adults has reached 11.6%. However, there is still no complete cure for diabetes at present, and the research on DM is also a common challenge faced by many scientists in the world. Basic research on type 2 diabetes (Type 2 Diabetes Mellitus, T2DM) or islet transplantation in type 1 diabetes (Type 1 Diabetes Mellitus, T1 DM) largely relies on research on animal islets, so successful isolation and purification of islets is a prerequisite for the above research. [0003] As early as 1911, some scholars extracted islets from the pancreas of guinea pigs, and then some scholars continued to improve this method,...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 李伟孙子林徐伟陈娟王晓航
Owner SOUTHEAST UNIV
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