Method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse

A technology of primary hepatocytes and transgenic mice, applied in the biological field, can solve the problems of less research and achieve the effect of high cell viability

Inactive Publication Date: 2018-12-21
THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse
  • Method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse
  • Method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Isolation and culture method of HBV transgenic mouse primary hepatocytes

[0084] ① Select suitable experimental mice (HBV transgenic mice) for the experiment, first disinfect the entire abdomen of the mice with 75% alcohol, and then inject 0.2ml of 1000units / ml heparin solution into the experimental mice by intraperitoneal injection After it is completely absorbed, inject 0.1ml of 2.5% pentobarbital sodium solution into its abdominal cavity to anesthetize the mouse, and finally fix the mouse on the operating board in the ultra-clean table (the whole process is aseptic operation) ready for use;

[0085] ②Use the sterilized ophthalmic scissors and ophthalmic forceps to carefully open the abdominal cavity of the mouse, dissect and free the upper and lower ends of the inferior vena cava close to the liver, and place a sterile surgical suture in the upper part of the liver of the inferior vena cava (for later ligation), and then use a new sterile indwelling needl...

Embodiment 2

[0089] Example 2 Identification method of HBV transgenic mouse primary hepatocytes

[0090] ① Detection of mouse primary hepatocyte albumin:

[0091] Firstly, the mouse primary hepatocytes were cultured for 24 hours, and then carefully washed with sterile PBS buffer (5min, repeated 3 times), the cell slides were taken out, and after drying, 5% paraformaldehyde was added dropwise. Make mouse primary hepatocytes fixed for 30min;

[0092] Then carefully wash the above-mentioned cell slides with sterile PBS buffer (5min, repeat 3 times), take out the cell slides and incubate them in a 3% filter prepared with sterile PBS after drying. hydrogen peroxide solution until no bubbles are produced in the cell slide;

[0093] Then carefully wash the above-mentioned cell slides with sterile PBS buffer (5min, repeat 3 times), take out the cell slides and incubate them in normal goat serum working solution (incubate at room temperature for 15 minutes) after drying. After discarding the wor...

Embodiment 3

[0105] Example 3 Morphological Identification Results of HBV Transgenic Mouse Primary Hepatocytes

[0106] The freshly isolated HBV transgenic mouse primary hepatocytes were inoculated on a cell culture dish. It can be seen under the microscope that most of the suspended HBV transgenic mouse primary hepatocytes are in the shape of crystal clear three-dimensional single balls, and a few aggregates can also be seen Arranged in clusters. After 3 hours, the primary hepatocytes of HBV transgenic mice have been uniformly dispersed and attached to the bottom of the cell culture dish. At this time, the typical structural characteristics of double nuclei of primary hepatocytes of HBV transgenic mice can be seen, and the cells are round or oval. 6 hours later, the double nuclei in the primary liver cells of HBV transgenic mice were more obvious, the intercellular space was larger, and the nuclei under a high-magnification microscope The kernels are clearly visible, the cells adhere mor...

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to a method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse. The method comprises the following steps: performing separating and collecting primary hepatocytes by an improved collagenase two-step perfusion method; filtering the collected hepatocytes by a sterile cell filter; centrifuging and adding a culture medium for resuspension; performing cell counting and inoculated culture; the identification includes mouse primary hepatocyte albumin detection, mouse primary hepatocyte glycogen detection and expression analysis of HBV in the mouse primary hepatocyte. According to the method provided by the invention, the obtained HBV transgenic mouse primary hepatocyte has high cell viability as well as the functions of parenchymal hepatic cells, and the functions can be maintained for 8 days so as to more truly reflect the physiological metabolic activity of the hepatocytes in a HBV infection state, which is of great significance to in-vitro study of the physiological metabolic function of the hepatocytes as well as to the study of antiviral drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for the isolation, cultivation and identification of HBV transgenic mouse primary hepatocytes. Background technique [0002] Hepatitis B infection is an infectious disease that seriously threatens human health and is the main cause of liver fibrosis and cirrhosis. Although the effective vaccination of hepatitis B vaccine this year has greatly reduced the incidence of hepatitis B, there are still many chronically infected people in the world. [0003] The treatment of chronic hepatitis B currently has no very effective antiviral specific drugs and therapies. Comprehensive treatments such as antiviral, immune regulation, improvement of liver function and anti-hepatic fibrosis are mainly used, among which antiviral therapy is the most important and key treatment measure. Currently clinical anti-HBV drugs mainly include interferons, nucleoside analogs, traditional Chinese medi...

Claims

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Application Information

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IPC IPC(8): C12N5/071G01N33/68C12Q1/02
CPCC12N5/067C12N2500/34C12N2509/00G01N33/5005G01N33/68
Inventor 李小松黄爱龙唐霓赵金秋王丹
Owner THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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