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Separating liquid for recess of pig intestinal tract and separating method thereof

A separation method and separation liquid technology, which can be applied to vertebrate cells, artificial cell constructs, animal cells, etc., can solve the problems of crypt structural integrity, purity and activity destruction, and achieve the effect of wide application value.

Active Publication Date: 2015-07-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to obtain porcine intestinal crypts with complete structure and high purity, and to provide materials for intestinal epithelial renewal research and tissue-specific transplantation, there have been studies on the isolation and purification of porcine intestinal crypts in the prior art. At present, various Evidence shows that porcine intestinal crypts can be isolated by EDTA chelation, however, the structural integrity, purity and viability of the crypts obtained by this method are somewhat compromised

Method used

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  • Separating liquid for recess of pig intestinal tract and separating method thereof
  • Separating liquid for recess of pig intestinal tract and separating method thereof
  • Separating liquid for recess of pig intestinal tract and separating method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Observation of crypt-villous axis structure

[0039] Anesthetize the 1-day-old Landrace piglet, cut the piglet’s abdomen open after bloodletting, take out the jejunum, wash the contents of the intestinal cavity with ice-cold HBSS, cut the jejunum longitudinally, and then use ice-cold HBSS to clean the intestinal epithelium. Thoroughly wash away the impurities on it; under a dissecting microscope, use tweezers to separate the muscle layer and epithelium of the jejunum, take a small amount of porcine intestinal epithelium and place it on a glass slide to make a pressed sheet and observe it under a microscope. The results are as follows: figure 1 .

Embodiment 2

[0040] Embodiment 2 Screening piglets of suitable age

[0041] This example involves the same method of killing piglets as in Example 1; select 1, 3, 5, 7 and 21 day-old piglets respectively, and get their jejunum to separate intestinal crypts by the following method:

[0042] (1) Take a 15cm jejunum and turn it into an intestinal sac, and wash the intestinal sac with HBSS;

[0043] (2) Ligate one end of the intestinal sac, pour the largest amount of separation solution 1 into the intestinal sac, and then ligate the other end;

[0044] (3) Put the intestinal capsule perfused with separation solution 1 into a 100mL Erlenmeyer flask, add 50mL separation solution 1 (30.0mM Na 2 EDTA-2H 2 O), incubate in an air shaker at 37°C, the shaker speed is 150r / min, and after incubation for 10-20min, the separated fluff is mainly;

[0045] (4) Replace with new separation solution 1 and incubate in an air shaker at 37°C with a rotation speed of 150r / min. After incubation for 15 to 20 minu...

Embodiment 3

[0048] The isolation method of embodiment 3 porcine intestinal crypts

[0049] This example relates to piglet killing method and embodiment 1 is identical.

[0050] 5-day-old Landrace piglets were selected, the jejunum was taken out, the contents were cleaned with HBSS, and the crypts were separated by three different methods. In order to achieve the purpose of providing sufficient nutrition for intestinal tissue and inhibiting crypt anoikis to obtain highly active crypts, the separation liquid used in this example is named separation liquid 2, and its formula composition is: 10.0mM HEPES; 30.0mM Na 2 EDTA-2H 2 O; 0.50mM DTT; 0.25mM BSA; 2.5mM D-glucose; 2.5mM L-glutamine; 0.50mM DL-β-hydroxybutyrate sodium; 10mM Y-27632; 2 PO 4 ; 138 mM NaCl; 8 mM NaCl 2 HPO 4 -7H 2 O.

[0051] Method 1: scraping mucous membrane method

[0052] Take a jejunum of about 10 cm, cut it longitudinally, wash away the impurities adhering to the intestinal epithelium with HBSS, scrape off ...

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Abstract

The invention belongs to the technical field of a separating liquid for a recess of a pig intestinal tract and a separating method thereof. The separating liquid comprises the following components with concentrations: 5.0-10.0mM HEPES, 5.0-30.0mM Na2EDTA-2H2O, 0.50-1.0mM DTT, 0.25-0.50mM BSA, 2.5-3.0mM glucose, 0.5-2.5mM glutamine, 0.50-1.0mM sodium DL-beta-hydroxybutyrate, 2-10mM Y-27632, 1.5-2.7mM KCl, 1.0-1.5mM KH2PO4, 130-138mM NaCl and 5-8 mM Na2HPO4-7H2O. Under a low temperature condition, the recess of the pig intestinal tract intact in structure and relatively high in activity and purity is obtained for the piglet small intestine by matching the separating liquid and HBSS.

Description

technical field [0001] The invention relates to the technical field of tissue separation, in particular to a separation liquid of porcine intestinal crypts and a separation method thereof. Background technique [0002] The intestine is the main site of nutrient absorption and the largest immune organ in mammals. The intestinal epithelium is renewed every 3 to 5 days on average, and it is the tissue with the fastest self-renewal rate in adult mammals. The small intestinal epithelium is composed of intestinal villus-crypt axis, and the villus extending to the intestinal lumen contains three types of mature Epithelial cells: absorbing cells (enterocyte), goblet cells (goblet cells) and endocrine cells (enteroendocrine cells), which can be divided into functional areas and proliferative areas; functional areas are composed of mature epithelial cells that do not have the ability to differentiate, which have different Functional cells together constitute the villi structure, amo...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 王修启黎相广高春起严会超傅厚龙陈明霞翟振亚范宏博
Owner SOUTH CHINA AGRI UNIV
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