WO2011050672A1

WO2011050672A1 - In-vitro cultivation model of hepatitis virus, construction method and applications thereof

Info

Publication number: WO2011050672A1
Application number: PCT/CN2010/077633
Authority: WO
Inventors: 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
Original assignee: 中国人民解放军军事医学科学院卫生学环境医学研究所
Priority date: 2009-10-27
Filing date: 2010-10-10
Publication date: 2011-05-05
Also published as: CN101696396A; CN101914489A

Abstract

In-vitro cultivation model of hepatitis virus, construction method and applications thereof are provided. The in-vitro cultivation model of hepatitis virus is constructed by adopting human fetal liver stem cells, the construction method thereof comprises: separating and culturing human fetal liver stem cells, identifying the human fetal liver stem cells by immunohistochemical methods, infecting human fetal liver stem cells with hepatitis virus serum, and validating whether the human fetal liver stem cells have been infected by the hepatitis virus and the virus is propagated and secreted persistently to the outside of the cells. As the human fetal liver stem cells adopted by the model can be infected by hepatitis virus and persistently secret hepatitis virus, and the human fetal liver stem cells can be sub-cultured, the model can be utilized to screen the drugs for resisting hepatitis virus and to evaluate the effect.

Description

In vitro culture model of hepatitis virus and its construction method and application

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.

Background technique

It is well known that hepatitis B virus (HBV) is one of the leading causes of liver disease. About 350 million people worldwide are chronic HBV carriers, which can cause severe cirrhosis and primary liver cancer. Viral hepatitis B has a wide range of epidemics and is highly contagious. It is one of the most common infectious diseases in the world. Once infected with hepatitis B virus, the liver will develop inflammatory lesions, and liver cells will be damaged. The harm, therefore, the study of the pathogenesis of HBV and its prevention and treatment methods are of great significance.

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. However, the established in vitro model of cell culture has different degrees of limitations, so there is still no suitable ideal model. For example, adult 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) is not produced in cell culture, and the level of virus replication is low. In addition, 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, however, 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.

Hepatitis C virus (HCV) is also a global epidemic. According to the World Health Organization, the infection rate of HCV in the population is about 3%. About 1.7 billion people worldwide are infected with HCV. 3. 50,000 cases. The national HCV sero-epidemiological survey conducted in China showed that the anti-HCV positive rate in the general population in China was 3.2%, and about 40 million people were infected with HCV. HCV is one of the main pathogens causing chronic hepatitis in humans and post-transfusion hepatitis. About 60% of patients with acute hepatitis C are converted to chronic, while 8-46% and 11-19% of chronic patients develop cirrhosis and liver. Cellular cancer.

Due to the low concentration of HCV in infected tissues and serum, the high variation of HCV, the lack of a stable HCV in vitro cell culture system and the ideal experimental animal model, further research on HCV is hindered. In addition to humans, chimpanzees are the only experimental animals currently reported to be infected with HCV, and because of their inconvenient source, they are costly and limit their use.

So far, interferon-based antiviral treatment has only cured about 30% of patients, and no satisfactory therapeutic drugs and effective vaccines have been found. Therefore, there is an urgent need to find effective ways to prevent and treat HCV infection. The key is to establish a stable cell model for viral replication and passage to gain insight into the life cycle and pathogenesis of hepatitis C virus.

The current research status of 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. 3 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 (HSC) 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.

Up to now, there have been no reports on the use of human fetal liver stem cells to construct HBV and HCV in vitro culture models.

Summary of the invention

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.

In order to solve the above technical problem, 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.

Preferably, 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.

Preferably, in the above-described hepatitis virus in vitro culture model, the human fetal liver stem cells are various types of cells having stem cell characteristics related to development and regeneration of liver embryos.

Preferably, in the above-mentioned hepatitis virus in vitro culture model, 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.

Preferably, the method for constructing the above-mentioned hepatitis virus in vitro culture model comprises the following steps:

I. Isolation and culture of human fetal liver stem cells:

(1) Separation and culture of fetal liver stem cells: Injecting collagenase in situ into the liver, peeling off the hepatocyte suspension, filtering one, fully dispersing the cells, centrifuging the cell suspension, discarding the supernatant, and precipitating the cells, suspending the cells with the medium. Once the cells are placed in a centrifuge tube, placed on ice, precipitated, and the cells are precipitated by adhesion of hepatic stem cells. The cell suspension is removed. The cells are pelleted by centrifugation. One cell is suspended in PBS solution. Percol l is divided into volume fractions by using PBS solution. 30% - 90% concentration gradient was centrifuged and the cells in the cell layer suspension precipitated between different Perco lls were inoculated into a plastic flask, and the medium was DMEM/F12 medium containing 10% fetal bovine serum. Incubate overnight in a 37 ° C, 5% C0 ₂ incubator, and observe the cell viability with freshly stained cells with 0.25% trypan blue staining;

(2) Culture and passage of fetal liver stem cells: After 18 hours of cell inoculation, the culture medium was replaced for the first time, and the non-adherent cells were discarded, and the liquid was changed once every 3-5 days. After two weeks, the number of cells grew in logarithm, forming a small cell clone. After four weeks, the small clones gradually increased, and the bottom of the bottle was covered. The mass fraction was 0.25% trypsin-digested cells, and passaged at a ratio of 1:2.

II. Immunohistochemical identification of human fetal liver stem cells: SP immunohistochemical kit was used to detect AFP, 0V_6, CK19, CD34, CK18 surface markers, and PBS was used as a negative control instead of each surface marker. The steps are as follows: After centrifugation, PBS suspends the cells and inoculates them on the coverslips. A ventilated air-dried PBS is fully washed. The antigen is heat-repaired. After washing with PBS, it is reddish-brown stained positive for a hematoxylin counterstain;

III. 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.

IV. Verify that the hepatitis virus has infected human fetal liver stem cells by one or more of the following methods:

(1). Quantitative PCR to detect virus titer of cell supernatant at different times; (2) . ELISA detection of viral antigen secretion in cell supernatants at different times: Detection of viral surface antigen or core antigen by cell virus supernatant at different times using hepatitis virus antigen detection kit;

(3) In situ hybridization to detect intracellular virus: The virus-infected cells were subjected to biotin (POD) and fluorescent probe labeling in situ hybridization on the third and seventh days after virus washing.

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.

Preferably, the hepatitis virus is a hepatitis B virus or a hepatitis C virus or a hepatitis D virus or a hepatitis E virus.

Preferably, in the method for constructing the above-described hepatitis virus in vitro culture model, the human fetal liver stem cells are various types of cells having stem cell characteristics related to liver embryo development and regeneration.

Preferably, in the method for constructing the above-described hepatitis virus in vitro culture model, 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.

Specifically, 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.

It is also possible to incubate the stably growing human fetal liver stem cells with the progeny hepatitis virus of the above-mentioned hepatitis virus in vitro culture model, and the other steps are the same as above, thereby performing screening and effect evaluation of the anti-hepatitis virus drug in vitro.

Preferably, 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.

Preferably, 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. Preferably, the above-mentioned application of the hepatitis virus in vitro culture model, the in vitro anti-hepatitis virus drug comprises a chemical drug, a Chinese herbal medicine, a bioengineering drug or an interferon.

The beneficial effects of the invention are:

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.

DRAWINGS

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 (ΙΟΟχ);

C is a schematic diagram of trypan blue staining (40χ);

D is a schematic diagram of HE staining (200 χ);

E is a schematic diagram of fetal liver stem cells (ΙΟΟχ) after 1 week of culture;

F is a schematic diagram of fetal liver stem cells (200 χ) 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 (ΙΟΟχ);

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

A is AFP; B is 0V-6; C is CD34; D is CK18; E is CK19; F is a negative control.

Figure 4 is a schematic representation of in situ hybridization (200 x) for detecting intracellular HBV DNA. among them

A and B are cells after infection for 3 days; C and D are cells after infection for 7 days;

E, F are negative controls; A, C, E are biotin labels;

B, D, and F are fluorescent labels.

Figure 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,

1.200 IU IFN; 2.500 IU IFN; 3.1000 IU IFN; 4. 2000 IU I FN; 5. Infected group control group; 6. Negative control;

M. DU000Marker.

Figure 6 is a flow chart showing the method of Embodiment 3 of the present invention.

detailed description

In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below with reference to the specific embodiments and the accompanying drawings.

Example 1 Construction of Hepatitis Virus in Vitro Culture Model As shown in Figure 1 and Figure I. Isolation and culture of human fetal liver stem cells:

(1) 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. . 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 μηι, the nucleus was round or oval, and it was in the shape of cobblestone (as shown in Figure 2Α, Β). 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. After 3-10 days of culture, the cell volume increased (as shown in Fig. 2E, F). The cell proliferation was obviously active, and the primary culture was continuously cultured. After two weeks, 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.

(2). Culture and passage of fetal liver stem cells: 10% FBS, 10 ng/ml SCF (human stem cell growth factor), 10 ng/ml HGF (human hepatocyte growth factor), 20 ng were replaced for the first time after 18 h of cell inoculation. /ml EGF (epidermal growth factor), 10 ng/ml LIF (human leukemia inhibitory factor) and 1 g/ml insulin in DMEM/F12 medium, and the unattached cells were discarded. Change the liquid every 3 to 5 days later. After two weeks, the number of cells grows logarithmically, forming small cell clones. After four weeks, the small clones gradually increase and spread the bottom of the bottle. Mass fraction 0. 25% trypsin digested cells and passaged at a ratio of 1:2.

II. Immunohistochemical identification of human fetal liver stem cells: SP immunohistochemistry kit was used to detect AFP, 0V_6, CK19, CD34, CK18 surface markers, and PBS instead of primary antibody as a negative control, the steps are as follows:

(1). After digesting the cells, centrifuge at low speed, suspend the cells in PBS, inoculate 100 μΐ, inoculate them on a cover glass, ventilate and dry, 95% alcohol at room temperature for 15 min;

(2) After PBS is fully washed, inactivated endogenous peroxidase by soaking in 30 ml/L hydrogen peroxide solution for 10 min;

( ³ ) After the antigen was heat-repaired, wash it with PBS three times, 5 min/time, and block with normal goat serum for 30 min at room temperature;

(4). Dry, add primary antibody (AFP, CK19, CD34, 0V-6) PBS 1: 100 diluted 4 °C overnight;

(5). Wash three times with PBS, 5 min / time, add biotin-labeled secondary antibody at 37 ° C for 45 min;

(6). Wash PBS three times, 5min / time, plus peroxidase streptavidin 37 ° C 45 min;

(7). Wash PBS three times, 5 min / time, add freshly prepared DAB, observe the red color staining positive for 5-10 min under the microscope;

(8) . Hematoxylin counterstaining, gradient ethanol dehydration, diphenyl benzene transparent, neutral gum seal. The color development results indicated that the newly isolated human fetal liver stem cells AFP, CK19, CK18, 0V-6,

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 μl 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.

IV. Verify that hepatitis B virus has infected human fetal liver stem cells and propagated and persisted to the extracellular:

Fluorescence quantitative PCR was used to detect viral titers of cell supernatants at different times.

(1) . 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.

(2) 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 37.7μ1; Taq enzyme 0·3μ1; UDG enzyme Ο. ΐμΐ; specimen solution or reference substance or standard 2μ1.

PCR reaction conditions: 37 ° C 5 min; 93 ° C 2 min; the following 40 cycles: 95 ° C 5 s;

60 ° C 40s.

The results showed that no virus was detected in the PBS washing solution at each sampling time point; 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;

In situ hybridization detection was performed on the third and seventh days after virus washing using human HBV DNA biotin labeling (POD) and fluorescence in situ hybridization double staining kit (Tianjin Yuyang Biotechnology Co., Ltd.) (according to kit instructions) The results showed that brown-red particles or green fluorescent signals were displayed in the cells of the infected virus, and the cell wells without the virus were negative, indicating that there were virus particles in the hepatitis B virus cells (as shown in Fig. 4A-F).

V. Verify that the model progeny HBV hepatitis virus is infectious 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.

Using the above-mentioned progeny HBV in vitro infection model, 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.

The above three methods were used to repeatedly verify that hepatitis B virus (including HBV high copy positive serum and this model progeny HBV hepatitis virus) has infected human fetal liver stem cells and propagated and continuously secreted outside the cell.

Example 2

I. 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.

II. Verify that hepatitis C virus has infected human fetal liver stem cells and propagated and persisted to the extracellular:

Detection of virus titer of cell supernatants at different times by real-time PCR

(1) RNA extraction: RNA 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.

(2) 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 20μ1.

PCR reaction conditions: 90 °C 30S; 61 °C 20min; 95 °C lmin; the following 50 cycles: 95 ° C 15 s; 60 ° C 60s.

The results showed that no virus was detected in the PBS washing solution at each sampling time point; HCV was high. 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. X 10 ⁵ copi es.

The ELI SA method detected positive HCeAg secretion by cells.

III. Verification of the progeny of this model HCV hepatitis virus is infectious

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.

Using the above-mentioned progeny HCV in vitro infection model, 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.

The other steps are the same as in the first embodiment.

Embodiment 3 As shown in Fig. 6, the difference from Embodiment 1 is as follows:

I. 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. Min. 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. 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 contents of the other steps are the same as those of the first embodiment.

Example 4 Screening and effect evaluation of anti-HBV drugs in vitro based on interferon I FN drugs

As shown in FIG. 5, using the HBV in vitro culture model described in Example 1, 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. No interferon group was used as positive control, no virus and interferon group were used as negative control, positive control, no virus and anti-HBV drug group as negative control, each group was repeated for three wells, incubated at 37 °C, and taken after 3 days. The supernatant, fluorescent quantitative PCR and electrophoresis methods were used to detect the inhibition of HBV reproduction.

The results showed that as shown in Fig. 5, 1-6, M: Interferon can inhibit the replication of HBV virus particles in cells, and different concentrations of interferon showed different inhibitory effects. This indicates that the HBV in vitro culture model was successfully constructed and can be used for anti-HBV drug screening and effect evaluation.

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. Incubate for 24 hours, aspirate the virus solution, wash 6 times with PBS, leave the wash solution for testing; add 200 IU/ml, 500 IU/ml, 1000 IU/ml, 2000 IU/ml dry 4 素 IFN, do not dry 4 Yousu group # positive control, no virus and interferon group as negative control, do positive control, no virus and anti-HCV drug group as negative control, each group repeated three wells, incubate at 37 °C, take 3 days later The supernatant, fluorescent quantitative PCR and electrophoresis methods were used to detect the inhibition of HCV reproduction.

The results showed that interferon can inhibit the replication of viral particles in cells. This demonstrates the successful construction of an HCV in vitro culture model and can be used for anti-HBV drug screening and effect evaluation.

Further, in 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.

In summary, 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.

The above detailed description of the hepatitis virus in vitro culture model, its construction method and application, with reference to the specific embodiments, are illustrative and not limiting, and several embodiments can be enumerated according to the scope of the limitation, so Variations and modifications of the general inventive concept are intended to be within the scope of the invention.

Claims

Claim

1. Hepatitis virus in vitro culture model, which is characterized by: It 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, and verification of hepatitis. The virus has infected human fetal liver stem cells and the virus has propagated and is continuously secreted outside the cell.

The in vitro culture model of hepatitis virus according to claim 1, wherein the hepatitis virus is hepatitis B virus or hepatitis C virus or hepatitis D virus or hepatitis E virus.

The in vitro culture model of hepatitis virus according to claim 1, wherein the human fetal liver stem cells are various types of cells having stem cell characteristics associated with liver embryo development and regeneration.

The in vitro culture model of hepatitis virus according to claim 3, wherein: said human fetal liver stem cells include oval cells, hepatocytes, facultative hepatocytes, small hepatocytes, hepatic side cells or pre-liver Somatic cells.

The method for constructing a hepatitis virus in vitro culture model according to claim 1, characterized in that: human fetal liver stem cells are used 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, and hepatitis. The virus serum infects human fetal liver stem cells, verifies that the hepatitis virus has infected the cells, and the virus has propagated and is continuously secreted outside the cells.

The method for constructing a hepatitis virus in vitro culture model according to claim 5, wherein the hepatitis virus is hepatitis B virus or hepatitis C virus or hepatitis D virus or hepatitis E virus.

7. The method for constructing a hepatitis virus in vitro culture model according to claim 5, wherein: said human fetal liver stem cells include oval cells, hepatocytes, facultative hepatocytes, small hepatocytes, and hepatic side cells. Or liver precursor cells.

The use of the hepatitis virus in vitro culture model according to claim 1, characterized in that the above-mentioned hepatitis virus in vitro culture model is used for screening and evaluating the anti-hepatitis virus drug in vitro.

9. The use of the hepatitis virus in vitro culture model according to claim 8, wherein: the human fetal liver stem cells in the model comprise oval cells, hepatocytes, and facultative liver Cells, small hepatocytes, hepatic side cells, or liver precursor cells.

The use of the hepatitis virus in vitro culture model according to claim 8, wherein the in vitro anti-hepatitis virus drug comprises a chemical drug, a Chinese herbal medicine, a bioengineering drug or an interferon.