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Method of separating pork liver stem cell

A stem cell and pig liver technology, applied in the field of pig liver stem cells, can solve the problems of low cell purity, small number of cells, long time required, etc., and achieve the effect of high purity and good survival rate

Inactive Publication Date: 2005-03-16
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The object of the present invention is to overcome the defect that the number of cells obtained by the separation method of the prior art is small, the cell purity is low or the required time is too long, thereby providing a method that can separate a sufficient amount in a short time, has high purity and a good survival rate. Method for isolating porcine liver stem cells

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  • Method of separating pork liver stem cell
  • Method of separating pork liver stem cell
  • Method of separating pork liver stem cell

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1, using the method of the present invention to isolate porcine liver stem cells

[0037] Experimental pigs underwent partial liver resection to promote liver regeneration. The steps are as follows:

[0038] 1) Use 20 mg / kg of pentobarbital sodium per kilogram of test pig body weight to carry out abdominal anesthesia to test pigs;

[0039] 2) Autoclave the surgical instruments used, and ultraviolet disinfection in the operating room;

[0040] 3) Cut off the hair on the abdominal cavity body surface of the test pig with electric clippers, disinfect the body surface of the test pig with 70wt% alcohol, and spread a towel;

[0041] 4) Cut the epidermis, and then dissect the abdominal cavity. Be careful not to cut the pleural membrane, so as not to cause death due to pneumothorax;

[0042] 5) Fix the epidermis and abdominal muscles with hemostats, add 0.90wt% normal saline dropwise into the abdominal cavity, push the mesentery to the left, add 0.90wt% normal salin...

Embodiment 2

[0061] Embodiment 2, identifying and analyzing porcine liver stem cells with flow cytometry (single staining detection)

[0062] Hepatic stem cells from 2 pigs were isolated according to the method in Example 1.

[0063] The porcine liver stem cells were identified by flow cytometry: freshly separated cells were added to eppendoff tubes (10 5 cells / tube), centrifuge at 1500rpm for 10min, wash once with PBS; loosen the cell pellet, add 1ml of cold acetone, fix at 4°C for 8min; centrifuge at 1500rpm for 5min, wash with PBS three times, and centrifuge at 1500rpm for 10min; Add 0.1ml PBS to make a cell suspension, add primary antibody albumin and CK-19 dropwise, and incubate at 37°C for 45 minutes; centrifuge at 1500rpm for 10 minutes, discard the supernatant, add 0.1ml PBS to the precipitate to make a cell suspension; Add PE-labeled IgG dropwise, incubate at 37°C for 30 minutes, wash with PBS 3 times; centrifuge at 1500rpm for 10 minutes, add 0.5-1ml PBS to the sediment dependin...

Embodiment 3

[0065] Embodiment 3, identifying and analyzing porcine liver stem cells with flow cytometry (double staining detection)

[0066] According to the method in Example 1, 6 days after partial hepatectomy, pig liver stem cells were isolated from 3 test pigs according to the method in Example 1, and each test pig obtained an average of 5.3 × 10 7 cells.

[0067] The double staining procedure is similar to the single staining in Example 2, except that the primary antibody is albumin + CK-19, and the secondary antibody includes FITC-labeled rabbit anti-mouse IgG (binding to CK-19) and PE-labeled goat antibody Rabbit IgG (albumin bound). Negative controls were replaced with FITC-labeled rabbit anti-mouse IgG and PE-labeled goat anti-rabbit IgG.

[0068] Biochemical phenotypes of freshly isolated porcine liver stem cells analyzed by flow cytometry, plotted in image 3 . The results of double staining showed that 92.23% of the cells co-expressed albumin and CK-19 (such as image 3 B...

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Abstract

The invention relates to a separating pork liver stem cell method, comprising steps of : cutting off a majority of liver to promote liver regeneration, adding collagenase to digest liver tissue into single cell, centrifuging to remove fibroblast of liver, using prolease E specificly digesting liver parenchymall cell , keeping the liver nonparenchymall cell, Percoll purifying using density gradient centrifugation method. The inventive method can provide highly purified, large amount and viable pork liver stem cell, which provide large amount of cells for stem cell fundamental research and clinical uses.

Description

technical field [0001] The invention relates to pig liver stem cells, in particular to a method for isolating pig liver stem cells. technical background [0002] Scientists predict that stem cell-related technologies may become a new method for treating refractory diseases in the 21st century. According to statistics, the total number of chronic hepatitis virus carriers in the world exceeds 350 million, of which more than 120 million are in my country. Clinically, acute liver injury, liver fibrosis, liver cirrhosis and even liver cancer caused by persistent infection of hepatitis B and C, alcohol or other poisons are more common, such as 20 million patients with chronic hepatitis B. The incidence of hepatitis and liver cirrhosis is increasing year by year, and thousands of liver disease patients die every year while waiting for liver transplantation. Liver transplantation is an effective means to solve advanced liver organic diseases, and the severe shortage of donor liver...

Claims

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Application Information

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IPC IPC(8): C12M3/00C12N5/074
Inventor 丰美福何祖平
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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