Establishment of hepatitis B virus (HBV) infection model under novel human liver biological scaffold three-dimensional culture system

A bio-scaffold, hepatitis B virus technology, applied in the field of biological research, can solve the problems of poor repeatability in vivo, huge phenotypic differences, low HBV infection efficiency, etc., and achieve the effect of high replication level, high infection efficiency and long infection duration.

Active Publication Date: 2019-04-26
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HBV stably integrated cell lines have played an active role in HBV drug screening and host-virus interaction research, but because the HBV genome has been integrated into the cell genome, such models cannot simulate the HBV infection process. In 2012, Li Wenhui's team first discovered Sodium ion / taurocholate cotransporting polypeptide (NTCP) is a functional receptor for hepatitis B and hepatitis D virus. This discovery has a profound impact on the research in the field of hepatitis B. By constructing a liver cancer cell that stably expresses NTCP The cell line can realize the natural infection of liver tumor cells to HBV, and is used for the research of HBV invasion mechanism and the screening of related drugs
However, liver tumor cells integrated with NTCP still face the problems of generally low HBV infection efficiency, and the phenotype of tumor cells is very different from that of normal liver cells, and the in vivo reproducibility is poor.

Method used

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  • Establishment of hepatitis B virus (HBV) infection model under novel human liver biological scaffold three-dimensional culture system
  • Establishment of hepatitis B virus (HBV) infection model under novel human liver biological scaffold three-dimensional culture system
  • Establishment of hepatitis B virus (HBV) infection model under novel human liver biological scaffold three-dimensional culture system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Preparation and identification of human liver cirrhosis ECM bioscaffold

[0091] 1. Experimental method

[0092] (1) Obtaining human cirrhotic liver tissue

[0093] Collected cirrhotic liver tissues from patients undergoing liver transplantation in Children's Hospital Affiliated to Chongqing Medical University, approved by the Ethics Committee of Children's Hospital Affiliated to Chongqing Medical University, and signed an informed consent form with the patients and their families. Harden the liver tissue 2-3 times, remove the blood cells and blood clots in the vessels, wash with PBS, and cut the liver into eight segments according to the anatomical structure of the liver tissue according to the Couinaud segmentation method. and the falciform ligament to segment the liver, cut it and place it in a -80°C refrigerator for cryogenic freezing and preservation.

[0094] (2) Preparation of human-derived liver cirrhosis ECM bioscaffold

[0095] 1. Cutting and stor...

Embodiment 2

[0122] In this example, a normal human-derived liver ECM bioscaffold was prepared.

[0123] 1. Experimental method

[0124] (1) Obtaining normal human liver tissue

[0125] The normal tissues adjacent to the cancer after liver tumor resection and discarded liver transplantation donor tissues were collected from the Children's Hospital Affiliated to Chongqing Medical University. They were approved by the Ethics Committee of the Children's Hospital Affiliated to Chongqing Medical University. The informed consent was signed with the patients and their families. The normal liver tissues during the operation After the body is isolated, lavage with heparin solution twice to remove blood cells and blood clots in the vasculature, and cut into small pieces with a size of about (5~7)mm*(5~7)mm*(5~7)mm. Blocks were placed in a -80°C refrigerator for low-temperature freezing and storage.

[0126] (2) The preparation method of normal human-derived liver ECM bioscaffold, comprising the fo...

Embodiment 3

[0143] The establishment of embodiment 3 hepatitis B virus infection model

[0144] 1. Test method

[0145] 1 Stereoscopic cell culture

[0146] 1.1. Disinfection of human-derived liver cirrhosis ECM bioscaffolds and normal human liver ECM bioscaffolds

[0147] Place the qualified human-derived liver cirrhosis ECM bioscaffold prepared in Example 1 and the qualified normal human-derived liver ECM bioscaffold prepared in Example 2 in 0.1% peracetic acid solution, and shake gently on a shaker for 30 minutes before replacing New 0.1% peracetic acid, shake lightly for 15 minutes, wash with sterile PBS solution for 10 minutes twice. Finally, it was sterilized under ultraviolet light for 2 hours.

[0148] 1.2. Three-dimensional cell culture

[0149] 1.2.1. Three-dimensional culture of primary hepatocytes

[0150] Human primary human hepatocytes (Primary Human Hepatocyte, PHH) were cultured in hepatocyte culture medium (Sciencell Company, Cat. No. 5210). Before inoculation, the c...

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Abstract

The invention belongs to the field of biological research and specifically relates to the establishment of a hepatitis B virus (HBV) infection model under a novel human liver biological scaffold three-dimensional culture system. According to the invention, the hepatitis B virus infection model is established through wide and deep research; the model has the main characteristics that the infectionefficiency is high, a viral load used in an infection process is much lower than that of other models, but the replication level of a virus in cells is high. Furthermore, the infection lasting time ofthe model is long, and specifically, the in vitro infection of common primary liver cells only can last for about 10 days, but the infection time of the model involved in the invention can last for nearly 20 days. In addition, the expression of the liver cells in the model involved in the invention is close to an in vivo real state and the model can be used for screening hepatitis B resisting medicines.

Description

technical field [0001] The invention belongs to the field of biological research, and in particular relates to the establishment of a hepatitis B virus infection model under a novel human liver biological scaffold three-dimensional culture system. Background technique [0002] Hepatitis B virus (HBV) infection is widespread worldwide and is one of the most serious diseases that endanger national health and cause national economic losses. Human hepatitis B virus has obvious species specificity and is only susceptible to primates such as gorillas and rhesus monkeys. In the cell model, only human primary liver cells can partially restore the HBV infection process to a certain extent, but the original The source of hepatocytes is difficult, and the ethical requirements are high; in vitro culture is easy to "dedifferentiate" and lose normal cell phenotype and function; at the same time, primary hepatocytes are expensive, so primary hepatocytes are not suitable for routine researc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/867C12Q1/02
CPCG01N33/5008C12N5/0671C12N15/86G01N2800/26C12N2730/10143
Inventor 张祯祯陈娟许红梅符州黄爱龙张明满
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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