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Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method

A technology of fetal liver cells and cells, which is applied in the field of medicine and biology, can solve the problems of difficult large-scale experiments and poor repeatability of experiments, and achieve good morphological characteristics of liver cells, long storage time, and large numbers of effects

Inactive Publication Date: 2014-04-23
康珞生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the above-mentioned limitations of PHHs, it is difficult to carry out experiments on a large scale, and the repeatability of experiments is poor.

Method used

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  • Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method
  • Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method
  • Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A method for separating, freezing and resuscitating human fetal liver cells of hepatitis B virus, the steps of which are as follows:

[0043] 1. Two-step collagenase perfusion method to separate human primary fetal liver cells:

[0044] The first step of the two-step collagenase perfusion method: the fresh liver tissue material is perfused with perfusion solution I for about 30 minutes through the exposed blood vessels until the blood in the liver tissue is washed away. The specific formula of perfusion solution I is: 8.00g / L sodium chloride, 0.40g / L potassium chloride, 3.57g / L hydroxyethylpiperazineethanesulfonic acid, 0.06g / L disodium hydrogen phosphate dihydrate, 0.06g / L potassium dihydrogen phosphate, 1g / L glucose, 1mM sodium pyruvate and 2mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, adjust the pH to 7.4, filter and sterilize with a 0.22μm microporous filter, and store at 4°C.

[0045] The second step of the two-step collagenase perfusion method: pe...

Embodiment 2

[0088] Using the cryopreservation, resuscitation and infection methods in Example 1, infection competition inhibition test, MyrB is a hepatitis B virus infection inhibitory small peptide, its protein sequence is: myristoylation-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG; before infection, different concentrations MyrB was pre-incubated with cells; during infection, a certain concentration of small peptides was still added to the infection medium. Such as Figure 4 C and Figure 4 D, MyrB inhibits HBV surface antigen secretion and intracellular core antigen expression in a dose-dependent manner.

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Abstract

The invention discloses a method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and an application of the method. The method comprises the following steps of thoroughly rinsing fresh hepatic tissues by utilizing a filling solution through an exposed blood vessel until complete digestion; placing the fresh separated hepatic cells into a culture dish containing cell washing liquid, and filtering the cells by utilizing a cell sieve to obtain single cell suspended liquid; precipitating washed single cells, re-suspending the cells, and storing the precipitated single cells to liquid nitrogen by utilizing a gradual freezing method; and establishing compact cell single-layer culture by optimizing planking density of high-vitality fetal hepatic cells, purifying the low-vitality hepatic cells through a Percoll centrifugal medium, subsequentially culturing the cells so as to resuscitate the fetal hepatic cells. By adopting the method, a great amount of fetal hepatic cells can be separated, stored and high-efficiently resuscitated, and the resuscitated fetal hepatic cells can maintain good characteristics of the hepatic cells, good differentiation state and good susceptibility to the hepatitis B virus, so that the problem that the primary hepatic cells are in shortage can be solved, and a stable hepatitis B virus medicinal sieve platform can be established.

Description

technical field [0001] The present invention relates to the field of medical biotechnology, in particular to a method for separating, freezing and resuscitating human fetal liver cells susceptible to hepatitis B virus and its application. The method can separate, freeze and resuscitate human fetal liver cells in large quantities, The resuscitated fetal liver cells maintained good liver cell function and susceptibility to hepatitis B virus. Background technique [0002] Human primary hepatocytes (abbreviated as PHHs) are currently considered to be the most ideal cell model for liver-specific pathological research in vitro. For example, using this kind of cells can establish in vitro infection of hepatitis B virus (abbreviated as HBV) . PHHs are also recognized as a valuable cell source for hepatocyte transplantation, bringing hope of rebirth to patients with liver failure. However, the scarcity of fresh liver tissue resources, limited number of hepatocytes, and differences ...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12Q1/70C12Q1/68A01N1/02G01N33/68
Inventor 胡康洪周明祁燕李家福成细瑶
Owner 康珞生物科技(武汉)有限公司
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