CN-122012374-A - Method for constructing mature liver organoid and HBV infection model of high expression NTCP

Abstract

The invention provides a method for constructing a mature liver organoid and HBV infection model with high expression of NTCP, which effectively improves the maturity of the liver organoid by adding 0.01 mu M-0.5 mu M of traditional Chinese medicine small molecule saikosaponin A (SSA) collaborative differentiation technology, and obtains the mature liver organoid with high expression of NTCP receptor. The HBV infection model established on the basis improves the invasion efficiency and replication level of viruses, and provides a reliable in vitro platform for researching the interaction between HBV and host cells and evaluating the curative effect of small molecules or antiviral drugs of traditional Chinese medicines.

Inventors

• WU FENFANG
• XU JUNMING
• WEI XIAFEI
• LI JINGYI
• FAN ZHONGYI
• LI NING
• CHEN XIAONI
• LIN SHAN
• HE FEI

Assignees

• 北京中医药大学深圳医院(龙岗)

Dates

Publication Date

20260512

Application Date

20260211

Claims (10)

1. 1. A method for constructing a mature liver organoid with high expression of NTCP is characterized by adding saikosaponin A with a final concentration of 0.01 mu M-0.5 mu M into early liver-like cell differentiation stage and maturation maintenance stage of human pluripotent stem cells induced and differentiated into the liver organoid, and culturing in a culture medium containing saikosaponin A for 5-10 days to finally obtain the mature liver organoid.
2. 2. The method for constructing mature liver organoids with high expression of NTCP according to claim 1, characterized in that the culture is performed in maturation maintenance stage using maturation medium comprising in addition to saikosaponin a, glutamine in a volume concentration of 2.5% -5%, 5ng/mL-20ng/mL of Oncoinhibin M and 0.2 μΜ -1.0 μΜ dexamethasone in HepatoZYME-SFM medium.
3. 3. The method of constructing a mature liver organoid highly expressing NTCP according to claim 1, characterized in that the concentration of saikosaponin a is 0.05 μΜ -0.1 μΜ.
4. 4. A method of constructing a model of HBV infection, comprising the steps of: I. obtaining a mature liver organoid using the method of any of claims 1-3; II. Pre-treating the mature liver organoids obtained in step I with a medium containing saikosaponin a to further stabilize the polar structure and maintain high levels of NTCP receptor expression; III, incubating HBV virus particles with the pretreated mature liver organoids to establish infection; IV, continuously culturing the infected mature liver organoids in a maintenance medium supporting HBV replication, and constructing an HBV infection model.
5. 5. The method for constructing HBV infection model according to claim 4, wherein the pretreatment in step II is performed for 12-48 hours, the concentration of saikosaponin A is 0.01 μm-0.5. Mu.M, and the culture medium used in the pretreatment is maturation medium.
6. 6. The method for constructing HBV infection model according to claim 4, wherein in step III, HBV viral particles are prepared by culturing a cell line stably secreting HBV, collecting the supernatant of the cell culture, filtering, and enriching by sucrose density gradient ultracentrifugation.
7. 7. The method for constructing HBV infection model according to claim 4, wherein in step III, HBV viral particles are infected at a dose of 1000-3000GE/cell under the condition of incubation at 37℃for 18-36 hours.
8. 8. The method for constructing HBV infection model according to claim 4, wherein in the step IV, the maintenance medium is a maturation medium containing no dexamethasone and contains saikosaponin A at a concentration of 0.01. Mu.M to 0.5. Mu.M, and the culture is performed in the maintenance medium for 7 to 20 days.
9. 9. A model of HBV infection constructed by the method of any of claims 4-8.
10. 10. Use of the HBV infection model of claim 9 in screening anti-HBV drugs.

Description

Method for constructing mature liver organoid and HBV infection model of high expression NTCP Technical Field The invention belongs to the field of biological research, and particularly relates to a method for constructing a mature liver organoid and HBV infection model with high expression of NTCP. Background Hepatitis b virus (HEPATITIS B VIRUS, HBV) infection is one of the most important chronic viral liver diseases worldwide. A large amount of epidemiological data shows that HBV persistent infection is closely related to the occurrence of chronic hepatitis, liver fibrosis progression, cirrhosis and hepatocellular carcinoma, and places a long and heavy burden on the public health system. Although vaccination and the use of partial antiviral drugs have reduced to some extent the level of new infections and viral replication, there is currently no curative strategy capable of completely clearing covalently closed circular DNA (cccDNA), and many chronically infected patients remain. Therefore, there is a need to deeply analyze HBV invasion, replication and host interaction mechanisms and develop novel intervention means based thereon, relying on infection models that are stable, reproducible and highly mimic human liver physiological states. However, HBV infection has significant host specificity, and can only efficiently infect humans and very few higher primates, such as chimpanzees, and conventional mice and most experimental animals are naturally insensitive to HBV, severely limiting the construction and application of in vivo models. Although animal models such as humanized liver mice, tree shrews and the like break through species barriers to a certain extent, the problems of long modeling period, high cost, large individual difference, strict ethical limitation and the like still exist, and the requirements of large-scale drug screening and mechanism research are difficult to meet. In the aspect of in vitro model, primary human liver cells are limited in source and easy to dedifferentiate in vitro culture, and common liver cancer cell lines are easy to obtain and expand, but have obvious differences from normal liver tissues in the aspects of metabolic functions, receptor expression, immune related pathways and the like, and can only partially reflect HBV infection process. These limitations make the construction of a model of human HBV infection closer to the physiological state of the human liver a critical issue to be addressed. The rise of organoid technology provides an important opportunity for the establishment of a new generation of humanized in vitro infection model. Liver organoids formed by three-dimensional culture of human pluripotent stem cells (hPSCs) or tissue-specific stem cells can form a structural unit similar to liver tissues in vitro by self-organization, express various liver marker genes and function-related enzymes, partially reproduce the characteristics of bile acid metabolism, drug metabolism, polarity differentiation and the like, and provide a brand-new experimental platform for researching liver development, liver disease occurrence and development and personalized medicine application. In the HBV research field, the liver organoids are expected to break through the multiple limitations of the traditional two-dimensional cells and animal models in the aspects of infection susceptibility, patient specific reaction, long-term culture stability and the like, and become an important bridge for connection basic research and transformation application. However, existing liver organoids still have significant drawbacks when used to construct HBV infection models. The liver-like cells obtained by most differentiation schemes have limited maturity, the overall function is more similar to that of fetal liver, and the expression level of drug metabolism zymogram, bile acid transport and key invasion receptors (such as NTCP and the like) is lower, so that natural susceptibility to HBV is low, and the infection efficiency and the virus replication level are lower. There is an urgent need to develop a new liver organoid model with both higher maturity and stronger HBV susceptibility, providing a reliable platform for subsequent mechanism research and antiviral drug screening. Disclosure of Invention In view of the above, the present invention aims to provide a method for constructing a mature liver organoid and HBV infection model with high expression of NTCP, which promotes the development and maturation of liver cells in the liver organoid through a small molecule saikosaponin A of traditional Chinese medicine, and remarkably improves HBV infection susceptibility and replication level while improving the organoid maturity, and on the basis, constructs a new HBV in vitro infection model. In order to achieve the above purpose, the technical scheme of the invention is realized as follows: A method for constructing mature liver organoid with high expression of NTCP includes such steps