WO2011050672A1 - Modèle de culture in vitro du virus de l'hépatite, procédé de construction et applications associés - Google Patents

Modèle de culture in vitro du virus de l'hépatite, procédé de construction et applications associés Download PDF

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WO2011050672A1
WO2011050672A1 PCT/CN2010/077633 CN2010077633W WO2011050672A1 WO 2011050672 A1 WO2011050672 A1 WO 2011050672A1 CN 2010077633 W CN2010077633 W CN 2010077633W WO 2011050672 A1 WO2011050672 A1 WO 2011050672A1
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virus
cells
hepatitis
stem cells
fetal liver
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PCT/CN2010/077633
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Chinese (zh)
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李君文
金敏
陈照立
邱志刚
谌志强
王新为
王景峰
郭向飞
王姝
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中国人民解放军军事医学科学院卫生学环境医学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24251—Methods of production or purification of viral material

Definitions

  • the invention relates to an in vitro culture model and a construction method and application thereof, in particular to a hepatitis virus in vitro culture model and a construction method and application thereof.
  • HBV hepatitis B virus
  • HBV culture in vitro and in vivo One of the important aspects of studying the biological characteristics of HBV, the pathogenesis of hepatitis B, the screening of anti-HBV drugs in vitro and its prevention and treatment methods is to establish a convenient and effective HBV culture model in vitro and in vivo. It is now used to study the animal culture in vivo model of HBV, mainly including primate, tree-sentence, duck and the most widely used transgenic mouse model and human-mouse chimeric liver model; the model cells include adult hepatocytes and fetal liver cells. HepRG cell line and HepG-2 based transgenic cells. Although the animal model can simulate the HBV infection process in vivo, there are still problems such as low expression level of HBV replication, interspecies differences and immunodeficiency.
  • hepatocytes are a type of terminally differentiated cells that cannot be subcultured and matured after hepatocyte plating.
  • Hepatocyte function such as albumin production ability, loss of typical polygonal morphology, and gradual decline in sensitivity to viruses, thus limiting its practical application; fetal liver cells are also restricted in application because they cannot be subcultured; HepRG cell line model Not only is it impossible to obtain intact HBV particles, but it is also difficult to remove the possible effects of additives on the HBV infection process; whereas in the most widely used HepG-2-based HepG2. 2.
  • 15 transgenic cells HBV is integrated into host cells.
  • the chromosome cannot be removed, so its replication mode is different from that of natural infection.
  • cccDNA replication intermediate
  • the level of virus replication is low.
  • the long-term stable integration state of HBV and transfected liver cancer cells is difficult to study.
  • the mechanism of cell transformation In addition, non-hepatogenic cells infected with HBV have also been explored, and more and more studies have shown that HBV not only infects liver-derived cells, but also infects other cells such as bone marrow stem cells, endothelial progenitor cells, placental trophoblast cells, etc.
  • Cells with stem cell characteristics may not be used to establish an ideal cell culture model because HBV cannot or replicate in these cells and these cells are non-hepatic-derived cells, and the mechanism of HBV infection may be different.
  • HCV Hepatitis C virus
  • HCV in vitro cell culture model is as follows: 1 Hepatic cell model, including primary hepatocytes and fetal liver cells, but because of its low level of replication, it is also unstable and cannot meet the requirements of drug screening research; Research for infection mechanisms. 2 HCV genome transfection model: including Huh-7 cells transfected with HCV JFH-1 RNA, fetal liver cells transfected with HCV RNA without adaptive mutation, but the defect is that only HCV 2a, la can be subcultured, secreted by cell culture. Viral particles are more homogenous and less infectious than animal experiments.
  • Extrahepatic cell models include human lymphocytes, fibroblasts, mononuclear leukemia cells, cholangiocarcinoma cells, T cell lines, PBMC, but whether the virus infects non-hepatocytes in a way and pathway It is unclear whether the biological characteristics of HCV, which is similar to in vivo infection of liver cells, and which have been cultured for a long time in non-hepatocytes, have changed.
  • Hepatic stem cells are hepatic stem cells with self-proliferation ability and multi-directional differentiation potential, which can differentiate into bile duct cells and differentiate into hepatocytes. It does not specifically refer to a certain kind of cells, but consists of various types of cells with stem cell characteristics related to liver embryo development and regeneration, such as fetal liver stem cells and hepatoblasts (the early embryonic development of the liver). Stem cells in the liver, which simultaneously express the hepatocyte phenotype and biliary cell phenotype, the latter two can further differentiate into mature hepatocytes and biliary cells), facultative hepatocytes and their daughter cells in adult liver Round cells, etc. As a source of cells for the treatment of liver-related diseases, hepatic stem cells play an important role in liver cell transplantation, tissue engineering and gene therapy.
  • the technical problem to be solved by the present invention is to provide a model for in vitro culture of hepatitis virus.
  • Another technical problem to be solved by the present invention is to provide a method for constructing the above-described hepatitis virus in vitro culture model.
  • Another technical problem to be solved by the present invention is to provide an application of the above-described hepatitis virus in vitro culture model.
  • the technical solution of the present invention is:
  • the in vitro culture model of hepatitis virus is constructed by using human fetal liver stem cells, including isolation and culture of human fetal liver stem cells, immunohistochemical identification of human fetal liver stem cells, hepatitis virus serum infection of human fetal liver stem cells, verification of hepatitis virus infected human fetal liver Stem cells and the virus has propagated.
  • the hepatitis virus is cultured in vitro, and the hepatitis virus is hepatitis B virus or hepatitis C virus or hepatitis D virus or hepatitis E virus.
  • the human fetal liver stem cells are various types of cells having stem cell characteristics related to development and regeneration of liver embryos.
  • the human fetal liver stem cells include oval cells, hepatocytes, facultative hepatocytes, small hepatocytes, hepatic side cells or liver precursors. Cell.
  • a method for constructing a hepatitis virus in vitro culture model, using human fetal liver stem cells for constructing a hepatitis virus in vitro culture model including isolation and culture of human fetal liver stem cells, identification of human fetal liver stem cells, hepatitis virus serum infection of human fetal liver stem cells, verification of hepatitis virus The cells are infected and the virus has propagated and is continuously secreted outside the cell.
  • the method for constructing the above-mentioned hepatitis virus in vitro culture model comprises the following steps:
  • Hepatitis virus high copy positive serum Infecting human fetal liver stem cells Hepatitis virus serum is inoculated into six-well plate fetal liver stem cells in serum-free medium; after incubation, the supernatant is aspirated, and the cells are cultured in culture medium every 48 hours. The hepatitis virus can be obtained by taking the supernatant.
  • the above three methods can be used to repeatedly verify that the hepatitis virus has infected human fetal liver stem cells and propagated and continuously secreted outside the cells.
  • the hepatitis virus is a hepatitis B virus or a hepatitis C virus or a hepatitis D virus or a hepatitis E virus.
  • the human fetal liver stem cells are various types of cells having stem cell characteristics related to liver embryo development and regeneration.
  • the human fetal liver stem cells include oval cells, hepatocytes, facultative hepatocytes, small hepatocytes, hepatic side cells or liver precursor cells.
  • the application of the hepatitis virus in vitro culture model uses the above-mentioned hepatitis virus in vitro culture model for the selection and effect evaluation of anti-hepatitis virus drugs in vitro.
  • the stable growth of human fetal liver stem cells was inoculated into a six-well plate for 24 hours, and then incubated with the hepatitis virus serum of the above-mentioned hepatitis virus in vitro culture model for 24 hours, and the virus solution was aspirated, washed 6 times with PBS, and left to be washed.
  • the antiviral drugs of 200 IU/ml, 500 IU/mK 1000 IU/mK 2000 IU/ml were added, and the anti-hepatitis virus group was used as a positive control, and the virus-free and anti-hepatitis virus group was used as a negative control.
  • Three wells were repeated in each group, incubated at 37 °C, and the supernatant was taken after 3 days.
  • the proliferation inhibition of hepatitis virus was detected by real-time PCR and electrophoresis.
  • the above-mentioned hepatitis virus in vitro culture model is used, wherein the human fetal liver stem cells are various types of cells having stem cell characteristics related to liver embryo development and regeneration.
  • the above-mentioned hepatitis virus in vitro culture model is used, wherein the human fetal liver stem cells include oval cells, hepatocytes, facultative hepatocytes, small hepatocytes, hepatic side cells or liver precursor cells.
  • the in vitro anti-hepatitis virus drug comprises a chemical drug, a Chinese herbal medicine, a bioengineering drug or an interferon.
  • the hepatitis virus in vitro culture model human fetal liver stem cells can infect hepatitis virus and continuously secrete hepatitis virus, and thus the model can be used for screening and effect evaluation of anti-hepatitis virus drugs in vitro, and the method is convenient and reproducible; Liver stem cells can be subcultured, thus greatly expanding their use.
  • Embodiment 1 is a schematic flow chart of a method according to Embodiment 1 of the present invention.
  • Figure 2 is a schematic diagram showing the cell morphology of human fetal liver stem cells at different culture time
  • A is a schematic diagram of freshly isolated fetal liver stem cells (40x);
  • B is a schematic diagram of freshly isolated fetal liver stem cells ( ⁇ );
  • C is a schematic diagram of trypan blue staining (40 ⁇ );
  • D is a schematic diagram of HE staining (200 ⁇ );
  • E is a schematic diagram of fetal liver stem cells ( ⁇ ) after 1 week of culture
  • F is a schematic diagram of fetal liver stem cells (200 ⁇ ) after 1 week of culture
  • G is a schematic diagram of fetal liver stem cells (100 ⁇ ) after 2 weeks of isolation and culture;
  • H is a schematic diagram of fetal liver stem cells (100 ⁇ ) after 4 weeks of isolation and culture;
  • I is a schematic diagram of subculture for 5 days of cells ( ⁇ );
  • J is a schematic diagram of cells (100 ⁇ ) after 2 weeks of passage
  • Figure 3 is a schematic representation of immunocytochemical staining (X 200 ) of different surface markers of human fetal liver stem cells. among them
  • Figure 4 is a schematic representation of in situ hybridization (200 x) for detecting intracellular HBV DNA. among them
  • E, F are negative controls; A, C, E are biotin labels;
  • B, D, and F are fluorescent labels.
  • FIG. 5 is a schematic diagram showing the electrophoresis of PCR products of HBV DNA after HBV treatment with different concentrations of interferon (IFN). among them,
  • Figure 6 is a flow chart showing the method of Embodiment 3 of the present invention.
  • Isolation and culture of fetal liver stem cells The collagenase was perfused into the liver in situ, and the perfusion was stopped when the liver was grayish white. The small cell scraper carefully peeled off the hepatocyte suspension, filtered through a 100 mesh screen, and the pipette was blown to fully spread the cells. . The cell suspension was centrifuged for 5 min at 4 °C with D_Hank, s solution 2000 r/min. The supernatant was discarded, and the precipitated cells were suspended in 2 ml of DMEM medium, and 50 ml of DMEM medium containing 0.1% Pronase E and 0.005% DNase I was added, and cultured at 37 ° C, 5% CO 2 for 30 min.
  • the cells were then transferred to a centrifuge tube and placed on ice for 20 min to precipitate the cells using hepatic stem cell adhesion.
  • the cell suspension was removed, and the pellet was centrifuged at 4 ° C, 2000 r / min for 10 min.
  • the cells were suspended in PBS.
  • Percol l was mixed into a concentration gradient of 30%-90% by volume in PBS, centrifuged at 4 °C, 10 000 r/min for 30 min, and the fine monthly coating between 50% and 70% Perco l 1 was taken up. .
  • DMEM/F12 suspension-precipitated cells 2 ml of DMEM/F12 suspension-precipitated cells were inoculated in 0.25% gelatin-treated plastic flasks, and the medium was DMEM/F12 medium containing 10% fetal bovine serum, and incubated at 37 ° C, 5% CO 2 Incubate overnight in the box. After the cells were inoculated for 18 hours, the DMEM/F12 medium containing 10% FBS, 10 ng/ml SCF, 10 ng/ml HGF, 20 ng/ml EGF, 10 ng/ml LIFf and 1 g/ml insulin was replaced for the first time. Adherent cells. Change the liquid every 3 to 5 days later.
  • the newly isolated stem cells were observed for cell viability with 0.25% trypan blue staining. Under the microscope, the cells were uniform in size, bright and transparent, with a large nuclear-to-cytoplasmic ratio. The cell diameter was 10-15 ⁇ , the nucleus was round or oval, and it was in the shape of cobblestone (as shown in Figure 2 ⁇ , ⁇ ). The average activity of Trypanblue stained cells The rate was 90% (as shown in Figure 2C), and the conventional HE stained nuclei were larger (as shown in Figure 2D), with less cytoplasm and uniform cell morphology. Continued culture showed that the cells grew relatively slowly, and the cells were in a semi-adherent state after inoculation for 24 hours.
  • the cell volume increased (as shown in Fig. 2E, F).
  • the cell proliferation was obviously active, and the primary culture was continuously cultured.
  • the cells grew as colonies and arranged in a grape shape (as shown in Figure 2G); after one month of culture, The cloning of the cells is increased, and the number of clones is gradually increased (as shown in Fig. 2H).
  • the trypsin digestion is 1:2 passage, and the cells are cloned and grown after passage (as shown in Fig. 2 I and J), and the cells gradually fall off with passage. , but still visible small clones.
  • CD34 was positive staining, in which fetal liver cell markers AFP and hepatic oval cell markers 0V-6 and CK18 were strongly expressed, positive cells showed brown-red cytoplasm, CD34 was also expressed, cytoplasm reddish brown staining was relatively small, and CK19 was positive. Cells stained weakly (as shown in Figures 3A-F). III.
  • HBV high copy positive serum infected human fetal liver stem cells HBV (10 7 -10 S Copies) serum 100 ⁇ l was inoculated into 1 ml DMEM/F12 medium (serum free) in six-well plate cells; after incubation at 37 ° C for 24 h The supernatant was aspirated, and the cells were cultured in DMEM/F12 medium (10% FBS). The supernatant was obtained every 48 hours, and the sample was continuously sampled 5 times.
  • Fluorescence quantitative PCR was used to detect viral titers of cell supernatants at different times.
  • DNA extraction DNA was extracted using the hepatitis B virus nucleic acid quantitative detection kit and stored at -20 ° C. The yin and yang control samples were used for the same treatment.
  • Fluorescence quantitative PCR amplification Fluorescence quantitative PCR amplification using the PCR-fluorescent probe method using the hepatitis B virus nucleic acid quantitative detection kit (Hangzhou Bori Company), the specific method of use can be found in the product manual.
  • PCR reaction system PCR reaction solution 37.7 ⁇ 1; Taq enzyme 0 ⁇ 3 ⁇ 1; UDG enzyme ⁇ . ⁇ ; specimen solution or reference substance or standard 2 ⁇ 1.
  • HBV high-copy serum can infect human fetal liver stem cells and can reproduce in the cells, and the cells can continuously secrete viral DNA to the supernatant for 10 days, and HBV The DNA content is between 10 2 _10 5 copies and the highest can reach 8.9 X 10 4 copies the next day.
  • ELISA was used to detect the secretion of viral HBsAg in cell supernatants at different times and in situ hybridization to detect intracellular virus distribution.
  • the results of ELISA in accordance with the kit instructions) using the hepatitis B virus s antigen detection kit (Intech Xinchuang Technology Co., Ltd.) showed that HBsAg was continuously detected in the cell supernatant;
  • the model progeny HBV hepatitis virus infected human fetal liver stem cells Infected human fetal liver stem cells: The model progeny HBV hepatitis virus (10 4 _10 5 Copies) was inoculated into 1 ml DMEM/F12 medium (serum free) six-well plate In the cells, after incubating at 37 ° C for 24 h, the supernatant was aspirated, and the cells were cultured in DMEM/F12 medium (10% FBS). The supernatant was obtained every 48 h, and the sample was continuously sampled 5 times.
  • DMEM/F12 medium serum free
  • the virus titer of cell supernatants at different times was detected by real-time quantitative PCR.
  • the results showed that the progeny HBV hepatitis virus can infect human fetal liver stem cells and can reproduce in cells, and the cells can be continuously secreted.
  • the virus reached the supernatant for 10 days, and the HBV DNA detection value was between 10 2 _10 5 copies.
  • the HBsAg secreted by the ELISA method was positive.
  • hepatitis B virus including HBV high copy positive serum and this model progeny HBV hepatitis virus
  • HCV high copy positive serum infected human fetal liver stem cells HCV (10 5 -10 7 Copies) serum lOOul was inoculated into 1 ml DMEM/F12 medium (serum free) in six-well plate cells; after incubation at 37 °C for 24 h The supernatant was aspirated, and the cells were cultured in DMEM/F12 medium (10% FBS). The supernatant was obtained every 48 hours, and the sample was continuously sampled 5 times.
  • RNA extraction was extracted using the Hepatitis C virus nucleic acid quantitative detection kit and stored at -20 ° C. The yin and yang control samples were used for the same treatment.
  • Fluorescence quantitative PCR amplification Fluorescence quantitative PCR amplification using the PCR-fluorescent probe method using the Hepatitis C virus nucleic acid quantitative detection kit (Hangzhou Bori Company), the specific method of use can be found in the product manual.
  • PCR reaction system RT-PCR MIX 25ul; Mn2+ 2.5ul; HCV Pribe MIX 1.5ul; internal Control lul; specimen solution or reference or standard 20 ⁇ 1.
  • PCR reaction conditions 90 °C 30S; 61 °C 20min; 95 °C lmin; the following 50 cycles: 95 ° C 15 s; 60 ° C 60s.
  • the sera can be infected with human fetal liver stem cells and can be propagated in the cells, and the cells can continue to secrete viral RNA to the supernatant for 10 days, and the HCV RNA content is between 10 2 _10 5 copies, and the highest energy can reach 5. 3 on the eighth day.
  • the ELI SA method detected positive HCeAg secretion by cells.
  • the model progeny HCV hepatitis virus infected human fetal liver stem cells The model progeny HCV hepatitis virus (10 5 Copies) l OOul was inoculated into 1 ml DMEM/F12 medium (serum-free) in six-well plate cells; 37 °C After incubation for 24 hours, the supernatant was aspirated, and the cells were cultured in DMEM/F12 medium (10% FBS). The supernatant was obtained every 48 hours, and the sample was continuously sampled 5 times.
  • the virus titer of cell supernatants at different time was detected by real-time PCR.
  • the results showed that the offspring HCV hepatitis virus can infect human fetal liver stem cells and can reproduce in cells, and the cells can be continuously secreted.
  • Viral RNA, HCV RNA content between 10 2 - 10 4 copies.
  • Embodiment 3 As shown in Fig. 6, the difference from Embodiment 1 is as follows:
  • Isolation and culture of fetal liver stem cells in human fetal liver stem cells The collagenase is perfused into the liver in situ, and the perfusion is stopped when the liver is grayish white. The small scraper is carefully peeled off the liver cell suspension, filtered through a 100 mesh screen, and pipette is blown. Allow the cells to spread out. The cell suspension was centrifuged at DV-Hank's 2000 r/min for 5 min at 4 °C. The supernatant was discarded, and the precipitated fines were suspended in 2 ml of DMEM medium, and 50 ml of DMEM medium containing 0.1% Pronase E and 0.005% DNase I, 37 ° C, 5% CO 2 culture 30 was added.
  • the cells were then transferred to a centrifuge tube and placed on ice for 20 min to precipitate the fine moon pack using hepatic stem cell adhesion.
  • the suspension of the fine moon was removed, and the pellet was centrifuged at 4 ° C and 2000 r/min for 10 min.
  • the cells were suspended in PBS.
  • Percol l was mixed into a concentration gradient of 30%-90% by volume in PBS, centrifuged at 4 °C, 10 000 r/min for 30 min, and the cell layer between 70% and 90% Perco ll was aspirated.
  • DMEM/F12 suspension-precipitated cells 2 ml of DMEM/F12 suspension-precipitated cells were seeded in 0.25% gelatin-treated plastic flasks in DMEM/F12 medium containing 10% fetal bovine serum at 37 ° C, 5% C02 incubator Cultivate overnight. After the cells were inoculated for 18 hours, the DMEM/F12 medium containing 10% FBS, 10 ng/ml SCF, 10 ng/ml HGF, 20 ng/ml EGF, 10 ng/ml LIFf and 1 g/ml insulin was replaced for the first time. Adherent cells. In the future, the liquid is changed every 3 to 5 days, and the stem cells are separated.
  • the selection and effect evaluation of the human fetal liver stem cells for the anti-HBV drug in vitro is to inoculate the stably growing human fetal liver stem cells into the six-well plate for 24 hours. Incubate it with HBV virus serum for 24 hours, aspirate the virus solution, wash 6 times with PBS, and leave the wash solution for examination; add interferon IFN at 200 IU/ml, 500 IU/ml, 1000 IU/mK 2000 IU/ml, respectively.
  • Example 5 In vitro anti-HCV drug screening and effect evaluation test based on interferon I FN drug
  • the HCV in vitro culture model described in Example 2, using human fetal liver stem cells for in vitro anti-HCV drug selection and effect evaluation is to stably grow human fetal liver stem cells in a six-well plate for 24 hours, and then with HCV virus serum.
  • Example 4 and Example 5 in addition to the interferon IFN drug, it may be a chemical drug or a Chinese herbal medicine or a bioengineering drug.
  • the human fetal liver stem cells used in the hepatitis virus in vitro culture model can be Infected with hepatitis B and C virus and persistent secretion of hepatitis B and C viruses; and human fetal liver stem cells can be subcultured; method of tube; good repeatability.

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Abstract

La présente invention concerne un modèle de culture in vitro du virus de l'hépatite, un procédé de construction et des applications associés. Le modèle de culture in vitro du virus de l'hépatite est construit en adoptant des cellules souches de foie fœtal humain, le procédé de construction de celui-ci comprenant les étapes consistant à séparer et à cultiver les cellules souches de foie fœtal humain, à identifier les celles souches de foie fœtal humain par des procédés immunohistochimiques, à infecter les cellules souches de foie fœtal humain par le sérum du virus de l'hépatite, et à valider si les cellules souches de foie fœtal humain ont bien été infectées par le virus de l'hépatite et si le virus s'est bien propagé et a bien été sécrété de manière persistante vers l'extérieur des cellules. Lorsque les cellules souches de foie fœtal humain adoptées par le modèle sont infectées par le virus de l'hépatite et sécrètent de manière persistante le virus de l'hépatite, et que les cellules souches de foie fœtal humain peuvent être sous-cultivées, le modèle peut être utilisé pour cribler des médicaments pour déterminer leur résistance au virus de l'hépatite et pour évaluer leur effet.
PCT/CN2010/077633 2009-10-27 2010-10-10 Modèle de culture in vitro du virus de l'hépatite, procédé de construction et applications associés WO2011050672A1 (fr)

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CN200910070982.0 2009-10-27
CN200910070982A CN101696396A (zh) 2009-10-27 2009-10-27 乙型肝炎病毒体外感染模型的构建方法及应用
CN201010245845.9 2010-08-05
CN 201010245845 CN101914489A (zh) 2009-10-27 2010-08-05 肝炎病毒体外培养模型及其构建方法与应用

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CN101696396A (zh) * 2009-10-27 2010-04-21 中国人民解放军军事医学科学院卫生学环境医学研究所 乙型肝炎病毒体外感染模型的构建方法及应用
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CN113583940A (zh) * 2021-08-31 2021-11-02 成都以邦医药科技有限公司 一种肝卵圆细胞永生化培养基及其制备方法和应用
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