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Method of activating lvier stem cell

A technology of liver stem cells and oval cells, applied in the field of activation of liver stem cells, can solve problems such as acetamidofluorene damage and liver parenchymal cell necrosis

Inactive Publication Date: 2004-11-24
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

However, this method can cause hepatic parenchymal cell necrosis, and acetamidofluorene (AAF) damages the central part of the liver lobules

Method used

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  • Method of activating lvier stem cell
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  • Method of activating lvier stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, using the method of the present invention to obtain activated liver stem cells

[0040] Ethiamin butyric acid was formulated as a 0.2 g / ml solution with double distilled water, and the amount of 0.08 wt% of the body weight of the rat was fed with ethaminobutyric acid every day for 6 days, and the animals were free to drink water and take Food;

[0041] For a major hepatectomy, the steps are as follows:

[0042] 1. the pentobarbital sodium of 40mg / kg mouse body weight is carried out intraperitoneal anesthesia to the mouse in step 1);

[0043] ② Ultraviolet disinfection in the operating room, using 70% alcohol to disinfect the body surface of the mouse;

[0044] ③Use sterile scissors and tweezers to cut the skin, and then use sterile scissors and tweezers to cut the abdominal cavity again. Be careful not to cut the pleural membrane, so as not to cause death due to pneumothorax;

[0045] ④Fix the skin and abdominal muscles with hemostatic forceps, add 0.90...

Embodiment 2

[0067] Embodiment 2, rat's liver, heart and kidney changes after subtotal hepatectomy 5 days

[0068] According to the method in Example 1, 10 rats were gavaged with ethylthioaminobutyric acid solution for 5 consecutive days, and 5 days after partial hepatectomy, the liver, heart, kidney and other tissues of the rats were randomly selected to be fixed with formaldehyde, dehydrated with alcohol, and then used Chloroform dealcoholization, paraffin-embedded sections, HE staining, microscopic examination.

[0069] Observing the changes of liver, heart and kidney in rats after partial hepatectomy 5 days under phase-contrast microscope, drawn in figure 2 , it can be seen from the figure that a large number of oval cells proliferate (as indicated by short arrows) around mature hepatocytes as indicated by long arrows. These oval cells are recognized as bipotential hepatic stem cells with oval nuclei and a large nucleoplasmic ratio. Moreover, no obvious pathological changes were see...

Embodiment 3

[0071] According to the method in Example 1, 15 rats were gavage-fed with ethylthioaminobutyric acid solution for 5 consecutive days. 2 / 3 part of the liver was removed, and 7 days after the operation, the oval cells of the liver were separated according to the method in Example 1:

[0072] Each rat obtained an average of 2.3 × 10 7 cells.

[0073] Freshly isolated cells were fixed with 2.5wt% cold glutaraldehyde for 30min, washed twice with PBS, fixed with 1wt% cold starvation acid for 4 hours, and then rinsed with PBS for 3 times; dehydrated with different concentrations of methanol, embedded in resin, ultrathin The sections were stained with uranyl acetate and lead citrate, and the ultrastructure of the cells was observed under a Philips EM 301 electron microscope ( image 3 ).

[0074] Observing the oval cells obtained in Example 3 under an electron microscope, it was found that the ultrastructural characteristics of the isolated cells were: ① oval nucleus; ② less cytopl...

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Abstract

The method of activating liver stem cell includes combined ethyl thioamino butyric acid and partial liver excision method, and conventional collagenase digesting and density gradient centrifugation method to separate oval liver cell. The method of the present invention can activate liver stem cell in short period without damage, and present invention makes it possible to provide great amount of cell for the preclinical research of liver stem cell and preclinical animal transplantation experiment.

Description

technical field [0001] The present invention relates to hepatic stem cells, in particular to a method for activating hepatic stem cells. technical background [0002] China is a big country with liver disease. At present, there are more than 20 million chronic hepatitis B patients, and more than 500,000 people die each year from advanced liver cirrhosis or liver cancer. Liver transplantation is an effective means to solve advanced liver organic diseases, but the biggest problem facing liver transplantation is the shortage of organ sources. Take the U.S., where there are relatively few liver diseases, as an example. About 18,500 people are ready to receive liver transplants every year, but less than a quarter of the patients are expected to receive donor liver transplants. In Asia, there are more than 100,000 patients with severe liver cirrhosis . One of the important ways to solve the severe shortage of donor liver organs is to carry out hepatic stem cell transplantation,...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/074
Inventor 丰美福何祖平汤越峰张好健
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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