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Liver precursor-like cell model derived from primary hepatocyte for hepatitis B virus infection, preparation method and application

A technology of primary liver cells and cell models, applied in the fields of liver precursor-like cells, hepatitis B virus infection research and its preparation, can solve the problems of inability to simulate HBV infection cell heterogeneity, exogenous gene introduction and high time cost , single genetic background of human liver cancer cells, etc., to achieve the effect of avoiding the risk of tumorigenesis, stable chromosomes, and high transformation efficiency

Active Publication Date: 2019-02-15
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above two conditions have the disadvantages of exogenous gene introduction and high time cost, and the genetic background of human liver cancer cell lines is single, which cannot simulate the heterogeneity of HBV-infected cells

Method used

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  • Liver precursor-like cell model derived from primary hepatocyte for hepatitis B virus infection, preparation method and application
  • Liver precursor-like cell model derived from primary hepatocyte for hepatitis B virus infection, preparation method and application
  • Liver precursor-like cell model derived from primary hepatocyte for hepatitis B virus infection, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Obtainment of Hepatic Precursor-like Cells HepLPCs and Identification of Related Characteristics

[0057] 1. Cell culture plate pre-coated

[0058] Use Matrigel matrix for coating, place the cryopreserved Matrigel matrix at 4°C overnight to make it liquid, dilute it with pre-cooled serum-free medium (such as DMEM) at 1:30, and add it to the culture well to cover the bottom surface. It can be used after placing it at 37°C for one hour.

[0059] 2. Obtaining HepLPCs from liver precursor-like cells

[0060] Take 2×10 4 cells / cm 2 Primary hepatocytes (PHCs) were inoculated in Matrigel-coated cell culture 6-well plates (purchased from Corning Company), and after adhering to the wall with WE medium containing 10% serum, the experimental group was replaced with improved hepatocytes The transformation medium (Transition and Expansion Medium, TEM) was cultured, and the control group was replaced with a commercially available hepatocyte growth medium (Hepatocyte gr...

Embodiment 2

[0073] Example 2 Three-dimensional culture and liver maturation of HepLPCs

[0074] 1. Three-dimensional sphere culture of HepLPCs

[0075] Will 2×10 6 A proliferating HepLPCs was digested into single cells in Trypsin TrypLE Express, and then resuspended in a mixed medium, which was prepared by mixing equal volumes of TEM medium and HMM medium, and then inoculated in a low-adhesion medium Cells were cultured in 6-well plates (purchased from Corning); after the cells formed spheres, the medium was changed to Hepatic Maturation Medium (HMM), and differentiation was continued in HMM medium for 8-10 days After further maturation, they become three-dimensionally differentiated functional hepatocytes (3D-Hep LPCs-Hep). During the cultivation process, photographs were taken and recorded with an optical microscope at different time points.

[0076] Hepatocyte differentiation medium HMM includes two parts: basic support and small molecule supplement. The basic support is DMEM / F12 me...

Embodiment 3

[0084] Example 3 3D-HepLPCs-Hep Infection with HBV and Application Research

[0085] 1. Preparation of HBV-infected cell model

[0086] 3D-HepLPCs-Hep was co-cultured with hepatitis B patient serum or HepG2.2.15 cell culture supernatant concentrate in the liver supplemented with 1% DMSO and 4% polyethylene glycol 8000. In the cell transformation and proliferation medium; after 24 hours of infection, use the medium to wash three to four times, then change the medium every 24 hours, collect the supernatant and store it in a -80°C environment for later use.

[0087] 2. Detection of HBV-related indicators

[0088] Take the supernatants collected above on the 2nd day, the 4th day, the 6th day, the 8th day, and the 10th day respectively, and use ABI 7500 (purchased from Life Technologies Corporation) real-time fluorescent PCR method to detect the HBV-DNA virus in the supernatant For titer, Architect i2000SR was used to detect HBeAg and HBsAg secreted in the supernatant through arc...

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Abstract

The invention belongs to the field of hepatitis B virus infected cell models in life sciences and medicine, and provides a liver precursor-like cell model derived from a primary hepatocyte for hepatitis B virus infection, a preparation method and application. The liver precursor-like cell model consists of functional liver cells after three-dimensional differentiation, and the functional liver cells after three-dimensional differentiation are obtained by liver precursor-like cells obtained after in vitro conversion and cultivation of human primary hepatocytes after three-dimensional cultivation and liver maturation cultivation. Through experimental verification, after the liver precursor-like cell model is infected with HBV, the related genes of HBV infections such as RXRA, HNF4A, NTCP andthe like can be expressed highly, and the cell model can be used in hepatitis B virus infection research or can be co-cultured with HBV for preparing a hepatitis B virus infection cell model.

Description

technical field [0001] The invention relates to the technical field of cell transformation, in particular to a kind of hepatocyte-derived Liver Progenitor-like Cells (HepLPCs) derived from human primary hepatocytes, and in particular to using the cells for three-dimensional culture in vitro for B The invention relates to research on hepatitis B virus (hepatitis B virus, HBV) infection and a preparation method thereof, belonging to the field of hepatitis B virus infection cell model in life science and medicine. Background technique [0002] Viral hepatitis type B (hepatitis B for short) is an infectious disease caused by HBV and mainly causes liver lesions. It is a global public health problem and one of the important infectious diseases in my country. Because the life cycle of HBV is relatively complex, it is of great significance to clearly understand the rules of HBV infection and replication for the prevention and treatment of HBV. In-depth medical research on the life ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/70C12Q1/02A61K31/7105A61P31/20A61K31/522
CPCA61P31/20G01N33/5014A61K31/522A61K31/7105C12N5/067C12Q1/04G01N2500/10C12N2501/15C12N2501/237C12N2501/405C12N2501/39C12N2501/415C12N2501/734C12N2513/00C12N2533/54C12N2533/90C12N2500/30C12N2500/38C12N2501/12C12N2501/11A61K2300/00
Inventor 鄢和新曾敏傅恭博周徐黄伟健吴红平王红阳
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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