CN107988260B - Method for establishing hepatitis B virus infected cell model by using pig primary hepatocytes and hNTCP recombinant lentiviruses - Google Patents
Method for establishing hepatitis B virus infected cell model by using pig primary hepatocytes and hNTCP recombinant lentiviruses Download PDFInfo
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Abstract
The invention discloses a method for establishing a hepatitis B virus infected cell model by utilizing porcine primary hepatocytes and hNTCP recombinant lentiviruses, belonging to the technical field of cell modification. Which comprises the following steps: step 1: preparing hNTCP recombinant lentivirus concentrated solution; step 2: preparing completely digested liver tissue; and step 3: preparing primary pig hepatocytes; and 4, step 4: preparing a porcine primary hepatocyte single cell suspension; and 5: preparing a cell suspension for plating; step 6: plating and culturing cells; and 7: carrying out hNTCP recombinant lentivirus infection on the primary hepatocytes of the pigs; and 8: and establishing a hepatitis B virus infected cell model. The invention constructs a recombinant lentivirus containing hNTCP gene in vitro, integrates the hNTCP gene into the genome of primary pig hepatocytes by the lentivirus with high infection efficiency, realizes the stable overexpression of hNTCP, and enables the primary pig hepatocytes to support hepatitis B infection.
Description
Technical Field
The invention relates to a method for establishing a hepatitis B virus infected cell model by utilizing porcine primary hepatocytes and hNTCP recombinant lentiviruses, belonging to the technical field of cell modification.
Background
Hepatitis b virus (abbreviated as HBV in english) infection is a "national disease" in china, and about 9000 ten thousand hepatitis b carriers in china and about one million people who die of hepatitis b infection-related diseases each year. The development of anti-HBV drugs is relatively delayed due to the lack of a suitable HBV infected cell model. Although hepatitis b vaccines can protect healthy people from hepatitis b, there is currently no curative drug for the large base of hepatitis b carriers. HBV infected cell models and animal models are of great importance for the development of anti-HBV drugs.
For a long time, cell models for anti-HBV drug screening and evaluation were limited to liver cancer cell lines such as HepG2, hepg2.2.15, Huh7, and the like. Since these cell lines are severely dedifferentiated and are far different in differentiation state from hepatocytes in vivo, the true antiviral effect and cytotoxic effect of the anti-HBV drug cannot be reflected in screening and evaluating the anti-HBV drug. Na (Na)+Taurocholate cotransport polypeptide (human Na)+hNTCP) is proved to be a hepatitis B receptor, and an HBV infected cell model is constructed based on liver cancer cell lines HepG2 and Huh7, and plays an important role in basic research of HBV and research and development of anti-HBV drugs. However, such cell lines have a relatively low differentiation state and are not effective in infection.
Human primary hepatocytes are recognized as "gold standard" in the development of anti-HBV drugs because they maintain the original state of the intra-hepatic environment. Human primary hepatocytes are the best model for research on HBV and research and development of antiviral drugs, but the human primary hepatocytes have the defects of limited sources, large batch differences, medical ethical problems and the like, and cannot be widely used in basic research on HBV and research and development of anti-HBV drugs. Experiments prove that the liver cancer cell lines from mice and rats still do not support HBV infection after the hNTCP is over-expressed, which shows that the high tropism and species specificity of HBV on human liver cells are proved, and other animal liver cells probably do not support HBV infection, thereby forming a prejudice.
Based on the close genetic distance between pigs and human, the similarity of liver transcriptome and the epidemiological investigation of pig serum, the hNTCP is over-expressed in primary hepatocytes of pigs, so that HBV infection is supported very likely, and a primary cell model and an animal infection model which are susceptible to HBV are prepared. Furthermore, efficient gene manipulation of primary hepatocytes is extremely difficult to achieve due to common transfection reagents such as Lipo2000 and electrotransfection. And lentivirus can realize high-efficiency infection and gene integration expression of primary hepatocytes. However, at present, a method for establishing a hepatitis B virus infected cell model by utilizing porcine primary hepatocytes and hNTCP recombinant lentiviruses does not exist.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for establishing a hepatitis B virus infected cell model by utilizing primary pig hepatocytes and hNTCP recombinant lentiviruses. The invention constructs a recombinant lentivirus containing hNTCP gene in vitro, integrates the hNTCP gene into the genome of primary pig hepatocytes by the lentivirus with high infection efficiency, realizes the stable overexpression of hNTCP, and enables the primary pig hepatocytes to support hepatitis B infection.
The technical scheme for solving the technical problems is as follows: a method for establishing a hepatitis B virus infected cell model by utilizing porcine primary hepatocytes and hNTCP recombinant lentiviruses comprises the following steps:
step 1: preparation of hNTCP recombinant lentivirus concentrate
Transfecting a lentivirus packaging plasmid pCMV dr8.91, a lentivirus packaging plasmid pMD.2G and an hNTCP overexpression plasmid pWPI-hNTCP-Puro into 293T cells simultaneously according to the mass ratio of 2:1:3 by using a calcium phosphate transfection method, collecting a culture supernatant containing lentiviruses 48h after transfection, and concentrating to obtain an hNTCP recombinant lentivirus concentrate;
step 2: preparation of well digested liver tissue
Digesting and separating primary pig hepatocytes by a two-step collagenase perfusion method to obtain completely digested liver tissues;
and step 3: preparation of porcine Primary hepatocytes
Transferring the completely digested liver tissue obtained in the step 2 to a culture dish containing cell washing liquid to obtain a primary pig hepatocyte cell suspension;
and 4, step 4: preparation of Single cell suspension of porcine Primary hepatocytes
Screening the porcine primary hepatocyte cell suspension obtained in the step 3 through a cell sieve to obtain a porcine primary hepatocyte single cell suspension;
and 5: preparation of cell suspension for plating
Centrifuging the single cell suspension obtained in the step 4, re-suspending with a cell washing solution, repeating the centrifugation for 3 times, and re-suspending the cells with a plating culture solution to obtain a cell suspension for plating;
step 6: cell plating and culture
The cell suspension for plating obtained in step 5 is mixed with (1.5-2).5)×105Viable cells/cm2Inoculating to collagen-coated culture plate or dish, adding plating culture solution, shaking, and culturing at 37 deg.C and 5% CO2After culturing for 4-6h in an incubator, replacing the plating culture solution with a hepatocyte maintenance culture medium PMM to obtain cultured primary porcine hepatocytes;
and 7: hNTCP recombinant lentivirus infection of primary hepatocytes of swine
Infecting the cultured primary porcine hepatocytes obtained in the step 6 with the hNTCP recombinant lentivirus concentrated solution obtained in the step 1 by using the infection number of 1 as an infection number, incubating for 24h, changing into a fresh hepatocyte maintenance culture medium PMM, changing for 1 time every 2 days, and detecting hNTCP protein expression on the 4 th day after infection to obtain hNTCP protein over-expressed primary porcine hepatocytes;
and 8: establishing hepatitis B virus infected cell model
And (3) adding a hepatitis B virus infection blocker into the hNTCP protein overexpressed pig primary hepatocytes obtained in the step (7), incubating for 30min, then infecting hepatitis B virus with the infection complex number of 1000, changing into a fresh hepatocyte maintenance culture medium PMM after infecting for 16-24h, changing for 1 time every 2 days, and detecting hepatitis B virus infection indexes on the 8 th day after infection to obtain a hepatitis B virus infected cell model established by using the pig primary hepatocytes and the hNTCP recombinant lentiviruses.
The invention proves that the primary pig hepatocytes support hepatitis B virus infection through lentivirus-mediated hNTCP overexpression and hepatitis B virus infection, has higher susceptibility to hepatitis B virus compared with a hNTCP stable-transfer hepatocellular carcinoma cell line, and can be used for anti-hepatitis B virus drug testing. In addition, compared with human primary hepatocytes, the porcine primary hepatocytes are not limited in source, small in difference between cell batches, free of medical ethical problems, and ideal cell models.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in step 1, the lentiviral packaging plasmid pCMV dr8.91, the lentiviral packaging plasmid pMD.2G and the hNTCP overexpression plasmid pWPI-Puro are all from the world plasmid-shared Addgene website. hNTCP overexpression plasmid pWPI-hNTCP-Puro hNTCP is cloned into pWPI-Puro vector by molecular cloning method. 293T cells, a cell line derived from human kidney epithelial cell line 293 cells by genetic technology, can express SV40 large T antigen, is a cell line commonly used for producing lentiviruses, and is purchased from China Center for Type Culture Collection (CCTCC for short).
Further, in step 1, the calcium phosphate transfection method specifically comprises: respectively adding 450 mu L of deionized water, 50 mu L of 2mol/L calcium chloride solution, 10 mu g of lentivirus packaging plasmid pCMV dr8.91, 5 mu g of lentivirus packaging plasmid pMD.2G and 15 mu g of hNTCP overexpression plasmid pWPI-hNTCP-Puro into a first centrifuge tube under the aseptic condition, and uniformly mixing to form a transfection solution I; adding 500 mu L of 2 XHBS solution into another centrifugal tube, shaking while dropping the transfection solution I to form a transfection solution II, and standing at normal temperature for 20 min; the transfection solution II was added to 293T cell culture dishes with a diameter of 10cm, a cell fusion degree of 70-80%, containing 10mL of DMEM transfection medium, at 37 ℃ with 5% CO2After the culture in the incubator for 12 hours, the DMEM transfection culture solution is changed into a fresh DMEM culture solution, namely the transfection is finished.
Further, the 2 XHBS solution was obtained by adding 8.00g of sodium chloride, 0.38g of potassium chloride, 0.10g of disodium hydrogen phosphate, 5.00g of hydroxyethylpiperazine ethanesulfonic acid, and 1.00g of glucose to 400mL of deionized water, adjusting the pH to 7.05, diluting to 500mL, performing filtration sterilization with a 0.22 μm filter, and storing at-20 ℃.
Further, the DMEM transfection medium is DMEM medium added with 10% v/v fetal bovine serum. Among them, DMEM medium and fetal bovine serum were purchased from GIBCO, USA.
The further beneficial effects of the adoption are as follows: DMEM transfection medium is antibiotic free.
Further, the fresh DMEM culture solution is prepared by adding 100units/mL of penicillin, 100mg/mL of streptomycin and 10% v/v of fetal calf serum to the DMEM culture solution.
Among them, DMEM medium, fetal bovine serum, penicillin and streptomycin were purchased from GIBCO, USA.
Further, in step 1, the concentration method comprises: putting the lentivirus supernatant into a 4 ℃ horizontal centrifuge, centrifuging for 10min at 3800 Xg/min, filtering with a 0.45-micron filter membrane, adding 15mL of filtrate into a 100KD ultrafiltration tube, transferring the 100KD ultrafiltration tube into the 4 ℃ horizontal centrifuge, centrifuging for 30min at 4000 Xg/min to obtain the hNTCP recombinant lentivirus concentrate.
The adoption of the further beneficial effects is as follows: concentration was divided into two centrifugations, the first centrifugation to initially remove dead cells or large cell debris. Filtration through a 0.45 μm filter was performed to remove small cell debris. And the second centrifugation is to cut off the molecules with molecular weight more than 100 daltons, including hNTCP recombinant lentivirus, so as to achieve the purpose of concentration.
The 100KD ultrafiltration tube was purchased from Millipore, USA.
Further, in the step 2, the two-step collagenase perfusion method is to perfuse fresh pig liver tissue with a perfusion solution I pre-cooled at 4 ℃ for 20min until blood in the liver tissue is washed clean, and perfuse the fresh pig liver tissue with a perfusion solution II pre-heated at 37 ℃ for 30min until the liver tissue loses elasticity.
Furthermore, the perfusion solution I is a mixed solution of 2mmol/l ethylene glycol bis (2-aminoethylether) tetraacetic acid in D-Hank's solution, the pH value is 7.4, the mixed solution is filtered by a 0.22 mu m filter membrane and is stored at 4 ℃.
Furthermore, the perfusion solution II is a mixture of D-Hank's solution containing 5mmol/l calcium chloride, 0.1g/l magnesium chloride hexahydrate, 0.3g/l collagenase type IV, 1.0g/l type II lyase and 50mg/l nuclease, and has a pH value of 7.4, and is filtered through a 0.22 μm filter membrane and stored at 4 ℃.
Among these, collagenase type IV was purchased from Sigma, USA. Type II isolation enzyme, Dispase II, was purchased from Roche, Switzerland. A nuclease, DNase I, was purchased from Solambio, China.
Furthermore, the D-Hank's solution contained 8.00g/l sodium chloride, 0.40g/l potassium chloride, 0.06g/l disodium hydrogenphosphate dihydrate, 0.06g/l potassium dihydrogenphosphate and 0.35g/l sodium hydrogencarbonate.
Further, in step 3, the cell washing solution is prepared by adding 100units/mL of penicillin, 100mg/mL of streptomycin and 10% v/v of fetal calf serum to a DMEM medium.
Further, in step 4, the pore size of the cell sieve is 70 μm.
Further, in step 5, the centrifugation conditions are all 50 Xg, 4 ℃, and the centrifugation time is 5 min.
Further, in step 5, the plating culture solution is prepared by adding 1% v/v ITS + premix, 2mmol/l glutamine, 10. mu.g/l epidermal growth factor, 18mg/l hydrocortisone, 40. mu.g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin and 5% v/v fetal calf serum to William's E culture medium.
Among them, William's E medium, WE for short, was purchased from GIBCO, USA. ITS + premix, available from GIBCO, USA. Epidermal growth factor, available from R & D, USA. Fetal bovine serum, purchased from GIBCO, usa.
Further, the ITS + premix is a 100-fold mother liquor containing 6.25. mu.g/mL insulin, 6.25. mu.g/mL transferrin, 6.25ng/mL selenious acid, 1.25mg/mL bovine serum albumin, and 5.35. mu.g/mL linoleic acid.
Further, in step 6, the method for preparing the collagen-coated culture plate or culture dish comprises: adding 172 mu L of glacial acetic acid into 100mL of deionized water, adding IV type rat tail collagen with the final concentration of 50 mu g/mL, filtering and sterilizing by using a 0.22 mu m filter membrane under the aseptic condition to obtain a collagen working solution, and keeping the collagen working solution at 4 ℃ for later use; the collagen working solution was added at a concentration of 5. mu.g/cm2The coating amount of the collagen is added into a culture plate or a culture dish which needs to be coated, after incubation for 1h at normal temperature, the collagen working solution is sucked out, naturally dried, sealed and preserved at 4 ℃.
Before the collagen-coated culture plate or dish is used, PBS is used for washing once to remove residual glacial acetic acid.
Further, in step 6, the hepatocyte maintenance medium PMM is prepared by adding 1% v/v ITS + premix, 2mmol/l glutamine, 10. mu.g/l epidermal growth factor, 18mg/l hydrocortisone, 40. mu.g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin and 2% v/v DMSO to William's E medium.
Among them, William's E medium, WE for short, was purchased from GIBCO, USA. ITS + premix, available from GIBCO, USA. Epidermal growth factor, available from R & D, USA. Fetal bovine serum, purchased from GIBCO, usa.
Further, the ITS + premix is a 100-fold mother liquor containing 6.25. mu.g/mL insulin, 6.25. mu.g/mL transferrin, 6.25ng/mL selenious acid, 1.25mg/mL bovine serum albumin, and 5.35. mu.g/mL linoleic acid.
Further, in step 8, the concentration of the blocking agent for hepatitis B virus infection is 500nmol/L, 50nmol/L, 5nmol/L, 0.5nmol/L, 0.05nmol/L, 0nmol/L, respectively, and the blocking agent for hepatitis B virus infection is Myr-PreS12-47。
The hepatitis B virus infection blocker Myr-PreS12-47Purchased from gill biochemistry (shanghai) ltd and having the amino acid sequence Myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG, wherein the amino terminus is Myristoyl (Myristoyl) modified.
Further, in step 8, the hepatitis B virus infection indexes are hepatitis B virus surface antigen, hepatitis B virus e antigen and intracellular hepatitis B virus core antigen in the supernatant.
The invention has the beneficial effects that:
(1) the lentivirus constructed by the invention can efficiently infect the primary porcine hepatocyte, integrates hNTCP into the genome, realizes the efficient and stable expression of the hNTCP, and simulates the original state of the primary human hepatocyte.
(2) The invention separates high-activity primary pig hepatocytes, plates the hepatocytes in a high-density mode, forms 100% fusion degree monolayer cell culture in a hepatocyte maintenance culture medium PMM, can maintain hepatocyte differentiation characteristics for about 1 month, and provides sufficient time guarantee for lentivirus infection, hNTCP expression, hepatitis B virus infection and subsequent virus replication and release.
(3) The invention establishes the hNTCP stably expressed pig liver primary cell, supports the high-efficiency infection of hepatitis B virus, has higher susceptibility and higher differentiation state compared with the hNTCP stably expressed liver cancer cell line, and is expected to replace the human primary liver cell to play a main role in hepatitis B virus basic research and anti-hepatitis B virus drug evaluation.
(4) Compared with human primary hepatocytes, the hNTCP stably-expressed porcine hepatocytes obtained by the invention have the advantages of unlimited sources, small cell batch difference, no medical ethics and the like; a novel in vitro cell model for screening and evaluating the anti-hepatitis B virus infection drugs is preliminarily established, so that the possibility is provided for developing an animal infection model with a complete immune system, and the method has important value for antiviral drug research.
Drawings
FIG. 1 is a plasmid map of recombinant lentivirus comprising hNTCP gene according to the present invention.
FIG. 2 is a cell morphology map of plated porcine primary hepatocytes cultured in accordance with the present invention at day 2.
FIG. 3 is a cell morphology map of plated porcine primary hepatocytes cultured in accordance with the present invention at day 30.
FIG. 4 is an immunofluorescence assay of hNTCP expression of the present invention versus a control of the localized human liver cancer cell line Huh 7D.
FIG. 5 is an immunofluorescence assay of hNTCP of the hNTCP expression and localization human liver cancer cell line Huh7D of the present invention.
Fig. 6 is an immunofluorescence assay of the control of hNTCP expression and localized porcine primary hepatocytes of the present invention.
FIG. 7 is an immunofluorescence assay of hNTCP expression and localized porcine primary hepatocytes of the present invention.
FIG. 8 is a graph showing the secretion test of e antigen of hepatitis B virus according to the present invention.
FIG. 9 is a view showing the secretion detection of hepatitis B virus s antigen according to the present invention.
FIG. 10 is a nuclear staining test chart of the hepatitis B virus infected human hepatoma cell line Huh7D of the present invention.
FIG. 11 is an immunofluorescence staining pattern for core antigen of hepatitis B virus of the hepatitis B virus infected human liver cancer cell line Huh7D of the present invention.
FIG. 12 is an overlay of nuclear staining of the hepatitis B virus infected human hepatoma cell line Huh7D and immunofluorescence staining of the core antigen of hepatitis B disease in accordance with the present invention.
FIG. 13 is a nuclear staining test chart of primary hepatocytes of hepatitis B virus-infected pigs according to the present invention.
FIG. 14 is the immunofluorescence staining pattern of the core antigen of hepatitis B virus of primary hepatocytes of swine infected with hepatitis B virus of the present invention.
FIG. 15 is an overlay of nuclear staining of primary hepatocytes of hepatitis B virus-infected pigs of the present invention and immunofluorescence staining of core antigens of hepatitis B disease.
FIG. 16 is a diagram showing the test and evaluation of the anti-invasion inhibitor of hepatitis B virus of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Examples
The method for establishing the hepatitis B virus infected cell model by utilizing the pig primary hepatocytes and the hNTCP recombinant lentiviruses comprises the following steps of:
step 1: preparation of hNTCP recombinant lentivirus concentrate
And (2) transfecting the lentivirus packaging plasmid pCMV dr8.91, the lentivirus packaging plasmid pMD.2G and the hNTCP overexpression plasmid pWPI-hNTCP-Puro into 293T cells simultaneously according to the mass ratio of 2:1:3 by using a calcium phosphate transfection method, collecting culture supernatant containing lentivirus 48h after transfection, and concentrating to obtain the hNTCP recombinant lentivirus concentrated solution.
(1) hNTCP expression plasmid construction
The lentiviral packaging plasmid pCMV dr8.91, the lentiviral packaging plasmid pMD.2G and the hNTCP overexpression plasmid pWPI-Puro are all from the world's plasmid-shared Addgene website (http:// www.addgene.org /). Obtaining human Na from human liver tissue+The gene coding sequence of the/taurocholate cotransporter polypeptide (hNTCP) is cloned into a pWPI-Puro empty vector by a molecular cloning method of enzyme digestion and linkage, and an hNTCP lentivirus expression plasmid pWPI-hNTCP-Puro is obtained (figure 1). 293T cells, a cell line derived from human kidney epithelial cell line 293 cells by genetic technology, can express SV40 large T antigen, is a cell line commonly used for producing lentiviruses, and is purchased from China Center for Type Culture Collection (CCTCC for short).
(2) Lentiviral production
Recovering 293T cells, culturing with fresh DMEM culture solution at 37 deg.C and 5% CO2And (5) culturing for 24 hours in an incubator. Cells were digested into single cells using trypsin, plated at 1:3 passages into cell culture dishes with a diameter of 10cm, cultured for 12h in DMEM transfection medium with a confluence of 293T cells of about 70-80%, and the plasmids were transfected into the cells by calcium phosphate transfection.
Fresh DMEM medium, DMEM medium (from GIBCO, USA) with 10% v/v fetal bovine serum (from GIBCO, USA), 100units/mL penicillin and 100mg/mL streptomycin. DMEM transfection medium was DMEM medium (purchased from GIBCO, USA) to which 10% v/v fetal bovine serum (purchased from GIBCO, USA) was added.
The calcium phosphate transfection method specifically comprises the following steps: respectively adding 450 mu L of deionized water, 50 mu L of 2mol/L calcium chloride solution, 10 mu g of lentivirus packaging plasmid pCMV dr8.91, 5 mu g of lentivirus packaging plasmid pMD.2G and 15 mu g of hNTCP overexpression plasmid pWPI-hNTCP-Puro into a first centrifuge tube under the aseptic condition, and uniformly mixing to form a transfection solution I; adding 500 μ L of 2 × HBS solution into another centrifuge tube, shaking while dropping transfection solution I to form transfection solution II, and standing at normal temperature for 20 min; the transfection solution II was added to 293T cell culture dishes with a diameter of 10cm, a cell fusion degree of 70-80%, containing 10mL of DMEM transfection medium, at 37 ℃ with 5% CO2After the culture in the incubator for 12 hours, the DMEM transfection culture solution is changed into a fresh DMEM culture solution, namely the transfection is finished.
A2 XHBS solution is obtained by adding 8.00g of sodium chloride, 0.38g of potassium chloride, 0.10g of disodium hydrogen phosphate, 5.00g of hydroxyethylpiperazine ethanesulfonic acid, and 1.00g of glucose to 400mL of deionized water, adjusting the pH value to 7.05, diluting to a constant volume of 500mL, filtering through a 0.22 μm filter for sterilization, and storing at-20 ℃.
DMEM transfection medium was DMEM medium (purchased from GIBCO, USA) to which 10% v/v fetal bovine serum (purchased from GIBCO, USA) was added.
Fresh DMEM medium, DMEM medium (from GIBCO, USA) was supplemented with 100units/mL penicillin (from GIBCO, USA), 100mg/mL streptomycin (from GIBCO, USA) and 10% v/v fetal bovine serum (from GIBCO, USA).
The concentration method comprises the following steps: putting lentivirus supernatant into a 4 ℃ horizontal centrifuge, centrifuging for 10min at 3800 Xg/min, filtering with a 0.45-micron filter membrane, adding 15mL of filtrate into a 100KD ultrafiltration tube (purchased from Millipore company, USA), transferring the 100KD ultrafiltration tube into the 4 ℃ horizontal centrifuge at 4000 Xg/min, centrifuging for 30min, taking out virus concentrate (about 250 mu L) retained in the 100KD ultrafiltration tube, and storing at-80 ℃ to obtain the hNTCP recombinant lentivirus concentrate.
(3) Lentiviral titer determination
Hepatoma cell line Huh7 cells at 5X 10 days before assay4Density of/well seeded in 96 well plates; carrying out 10-fold gradient dilution on the hNTCP recombinant lentivirus concentrated solution by using a DMEM culture solution containing 6 mu g/ml hexadimethrine bromide to respectively obtain dilution times of 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9The diluent of (4); sucking out original DMEM culture solution from 96-well plate, adding diluted virus infection solution into each well, standing at 37 deg.C and 5% CO2After overnight culture in an incubator, changing to 100 mu L of fresh DMEM culture solution, continuing to culture for 48h, carrying out trypsinization on each infected cell well in a 96-well plate at a ratio of 1:2 to 2 new culture wells, wherein puromycin containing 5 mu g/mL is added into 1 well, and blank control containing 1 well is added; and culturing for 48h, respectively calculating the number of cells in the puromycin treatment hole and the blank control hole in each dilution ratio through cell counting, and calculating the virus infection efficiency and the virus titer.
The virus infection efficiency was calculated as (number of cells in puromycin-treated well/number of cells in blank control well) × 100%, and the virus titer was calculated from the corresponding well having an infection efficiency of about 10%, where the virus titer (titer/mL) ═ virus infection efficiency × number of cells in puromycin-free-treated well × dilution factor.
Step 2: preparation of well digested liver tissue
The primary pig hepatocytes are digested and separated by a two-step collagenase perfusion method to obtain completely digested liver tissues.
Wherein, the two-step collagenase perfusion method comprises the following steps: fresh pig liver tissue was perfused through the exposed blood vessels with 4 ℃ pre-cooled perfusion solution I for 20min until the blood in the liver tissue was washed clean. Wherein the perfusion solution I is a mixed solution containing 2mmol/l ethylene glycol bis (2-aminoethylether) tetraacetic acid in D-Hank's solution, the pH value is 7.4, the mixed solution is filtered by a 0.22 mu m filter membrane and is stored at 4 ℃. The D-Hank's solution contains 8.00g/l sodium chloride, 0.40g/l potassium chloride, 0.06g/l disodium hydrogen phosphate dihydrate, 0.06g/l potassium dihydrogen phosphate and 0.35g/l sodium bicarbonate
The second step of the two-step collagenase perfusion method: perfusing with perfusion solution II preheated at 37 deg.C for 30min until the liver tissue of pig loses elasticity. Wherein the perfusion solution II is a mixed solution of D-Hank's solution containing 5mmol/l calcium chloride, 0.1g/l magnesium chloride hexahydrate, 0.3g/l collagenase type IV (purchased from Sigma, USA), 1.0g/l type II separating enzyme (Dispase II, purchased from Roche, Switzerland) and 50mg/l nuclease (DNase I, purchased from Solarbio, China), the pH value is 7.4, the mixture is filtered by a 0.22 mu m filter membrane, and the mixture is stored at 4 ℃.
And step 3: preparation of porcine Primary hepatocytes
And (3) transferring the completely digested liver tissue obtained in the step (2) to a culture dish containing a cell washing solution, and carefully shaking off the digested cells to obtain a primary porcine hepatocyte cell suspension.
Among them, the cell wash was DMEM medium (purchased from GIBCO, USA) to which 100units/mL of penicillin, 100mg/mL of streptomycin and 10% v/v of fetal bovine serum (purchased from GIBCO, USA) were added.
And 4, step 4: preparation of Single cell suspension of porcine Primary hepatocytes
And (4) screening the porcine primary hepatocyte cell suspension obtained in the step (3) through a cell sieve with the aperture of 70 mu m to obtain the porcine primary hepatocyte single cell suspension.
And 5: preparation of cell suspension for plating
And (4) centrifuging the single cell suspension obtained in the step (4), wherein the centrifugation conditions are as follows: 50 Xg, 4 ℃ for 5 min. After resuspension with cell wash, centrifugation was repeated 3 times, and cells were resuspended in plating medium to obtain plating cell suspension.
Wherein, the plating culture solution is prepared by adding 1% v/v ITS + premix (WE, purchased from GIBCO, USA) into William's E culture medium (WE for short), 2mmol/l glutamine, 10 μ g/l epidermal growth factor (purchased from R & D, USA), 18mg/l hydrocortisone, 40 μ g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin, and 5% v/v fetal calf serum (purchased from GIBCO, USA).
ITS + premix is 100-fold mother liquor, and contains 6.25. mu.g/mL insulin, 6.25. mu.g/mL transferrin, 6.25ng/mL selenious acid, 1.25mg/mL bovine serum albumin, and 5.35. mu.g/mL linoleic acid.
The cell density and cell viability of the cell suspension for plating are calculated, namely, after the cell suspension for plating and 0.4% m/v dolol blue staining solution are mixed uniformly according to the volume ratio of 1:1, mixed liquor is obtained, 20 mu L of the mixed liquor is rapidly added into a counting plate (purchased from Counterstar company, USA), and the numerical values of the cell density and the cell viability are read by a counter.
The preparation method of the 0.4% m/v Taiwan phenol blue staining solution comprises the following steps: 0.4g of dolichol blue solid was completely dissolved in 100mL of a phosphate buffer solution at a pH of 7.2 to 7.4, autoclaved, and kept at 4 ℃. A phosphate buffer solution, abbreviated in English to PBS, containing 8.0g/l of sodium chloride, 0.2g/l of potassium chloride, 3.58g/l of disodium hydrogen phosphate dodecahydrate and 0.24g/l of potassium dihydrogen phosphate.
Step 6: cell plating and culture
The cell suspension for plating obtained in step 5 was mixed at (1.5-2.5). times.105Viable cells/cm2Inoculating to collagen-coated culture plate or dish, adding plating culture solution, shaking, and culturing at 37 deg.C and 5% CO2Culturing for 4-6h in an incubator to allow the cells to adhere to the wall sufficiently, changing the plating culture solution into a hepatocyte maintenance culture medium PMM, culturing for 30 days, and recording the cell morphology by a phase contrast microscope on the 2 nd day and the 30 th day after plating respectively to obtain the cultured primary porcine hepatocytes.
The preparation method of the collagen-coated culture plate or culture dish comprises the following steps: adding 172 μ L glacial acetic acid into 100mL deionized water, adding IV type rat tail collagen with final concentration of 50 μ g/mL, and sterilizingFiltering and sterilizing with 0.22 μm filter membrane to obtain collagen working solution, and keeping at 4 deg.C; the collagen working solution was added at a concentration of 5. mu.g/cm2The coating amount of the collagen is added into a culture plate or a culture dish which needs to be coated, after incubation for 1h at normal temperature, the collagen working solution is sucked out, naturally dried, sealed and preserved at 4 ℃. The collagen-coated plates or dishes were washed once with PBS to remove residual glacial acetic acid before use.
Hepatocyte maintenance medium PMM was prepared by adding 1% v/v ITS + premix, 2mmol/l glutamine, 10. mu.g/l epidermal growth factor, 18mg/l hydrocortisone, 40. mu.g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin and 2% v/v DMSO to William's E medium.
And 7: hNTCP recombinant lentivirus infection of primary hepatocytes of swine
And (3) infecting the primary pig hepatocytes cultured in the step (2) of the step (6) with the infection number of 1 in the concentrated hNTCP solution obtained in the step (1), incubating for 24h, changing into a fresh hepatocyte maintenance culture medium PMM, changing for 1 time every 2 days, detecting the expression and positioning of hNTCP protein by an immunofluorescence method after fixing of a part of cells on the 4 th day after infection, and testing the infection of hepatitis B virus by other cells to obtain the primary pig hepatocytes over-expressed by the hNTCP protein.
And 8: establishing hepatitis B virus infected cell model
Adding hepatitis B virus infection blocker Myr-PreS1 with concentration of 500nmol/L, 50nmol/L, 5nmol/L, 0.5nmol/L, 0.05nmol/L and 0nmol/L into the hNTCP protein over-expressed pig primary hepatocytes obtained in step 72 -47(purchased from Jier Biochemical (Shanghai) Co., Ltd.), incubating for 30min, infecting hepatitis B virus in a hepatocyte maintenance culture medium PMM containing 4% w/v polyethylene glycol 8000 with the infection complex number of 1000, changing into a fresh hepatocyte maintenance culture medium PMM after infecting for 16-24h, changing for 1 time every 2 days, detecting hepatitis B virus infection index hepatitis B virus surface antigen (English abbreviation is HBsAg), hepatitis B virus e antigen (English abbreviation is HBeAg) and intracellular hepatitis B virus core antigen on 8 days after infecting, and obtaining the model for establishing the hepatitis B virus infected cell by utilizing the primary pig hepatocytes and the hNTCP recombinant lentivirus.
Detection and analysis
1. Detection of isolated primary porcine hepatocyte morphology and hNTCP expression
On one hand, morphologically observing the morphology of the primary porcine hepatocytes at different culture time points by using a phase contrast microscope; in another aspect, the expression and localization of hNTCP is identified using immunofluorescence.
The specific method of hNTCP immunofluorescence is as follows: taking 1 well of a 12-well plate as an example, when cells are cultured for 2 months, absorbing old culture solution, washing with PBS at normal temperature for 1 time, immediately washing with ice-cold 4% paraformaldehyde for 1mL (the specific formula is that 4g paraformaldehyde is weighed and placed in a triangular flask, 80mL deionized water is added, the flask is placed in a 37 ℃ constant temperature water bath box, shaking and uniformly mixing every 1-2h, after 16-24h, the paraformaldehyde is completely dissolved, supplementing deionized water to 100mL, adjusting the pH value to 7.2), and fixing at normal temperature for 10-15 min; washing with PBS on a horizontal shaker for 3 times, 5min each time; to enhance binding of the antibody to intracellular proteins, 0.5mL of PBS containing 0.25% v/v Triton100 was treated at room temperature for 45 min; washing with PBS on a horizontal shaker for 3 times, 5min each time; blocking, adding a proper amount of blocking solution (PBS solution containing 5% v/v goat normal serum and 2% m/v BSA), and horizontally shaking at normal temperature for incubation for at least 1 h; diluting the antibody with PBS solution containing 3% v/v goat normal serum, wherein the dilution ratio of the anti-hNTCP rabbit polyclonal antibody (purchased from Sigma company in the United states) is 1:500, and incubating overnight at 4 ℃ in a shaking table; washing with PBS solution containing 3% goat normal serum for 3 times (10 min each time) on a horizontal shaker to remove non-specifically bound antibody; the secondary antibody is goat anti-rabbit lgG coupled DyLight-488 (purchased from Thermo Fisher Scientific Inc., USA) and is diluted to PBS solution containing 3% of goat normal serum at a ratio of 1:1000, and the solution is incubated in dark for 1h at normal temperature, and then uniformly protected from light; washing with PBS solution containing 3% goat normal serum for 2 times (10 min each) on horizontal shaker, adding 1:1000 DAPI (from Roche, Switzerland) for staining cell nucleus during the third washing, and incubating at room temperature for 10 min; washing again to remove residual DAPI for 10-20 min; an appropriate amount of PBS solution containing 3% goat normal serum was added to prevent the cells from being deformed by drying during observation. Finally, the fluorescence signal was observed using a Leica DFC425C fluorescence microscope (available from Leica, Inc., Germany).
Analysis of results
(1) Morphological observation and analysis of primary porcine hepatocytes at different culture time points
The primary porcine hepatocytes separated by the two-step collagenase perfusion method show obvious differentiated hepatocyte morphology under the culture conditions of the invention. On day 2 after plating (fig. 2), the porcine primary hepatocytes were polygonal, round and highlighted in nuclei, abundant in organelles; at day 30 after plating (FIG. 3), the hepatocyte status was still good, the porcine cells were slightly aged and cell debris was significantly increased relative to day 2. Since the culture time of 30 days has satisfied the time period required for the infection with lentivirus, the infection with hepatitis B virus, the drug test, etc., the present inventors determined the time point as the end point of the culture. The picture scale is 150 μm.
(2) Immunofluorescence assay for hNTCP expression and localization
The expression and the location of the receptor hNTCP protein in the pig cells are confirmed by immunofluorescence by taking the human hepatoma cell line Huh7D as a control. As shown in fig. 4 to fig. 7, in the human hepatoma cell line Huh7D, only lentivirus-infected cells expressed the hNTCP protein, and the hNTCP protein was mainly localized on the cell membrane. Likewise, only lentivirus-infected porcine primary hepatocytes express the hNTCP protein, and the hNTCP protein is predominantly localized to the cell membrane. The hNTCP protein can be expressed in the primary porcine hepatocytes and is positioned correctly, and a foundation is laid for the hNTCP protein to exert biological functions. The picture scale is 150 μm.
2. HBV infection of hNTCP-expressing pig primary hepatocytes and anti-HBV invasion drug test
Method for detecting key indexes after virus infection by ELISA and immunofluorescence detection
The HBeAg detection refers to the instruction of hepatitis B virus e antigen diagnostic kit provided by Shanghai Korea company. Firstly, diluting a 25-fold concentrated washing solution provided in the kit to a working concentration of 1-fold, wherein the specific method comprises the following steps: 480mL of purified water is measured, 20mL of concentrated washing solution is added, and after the concentrated washing solution is fully and uniformly mixed, 500mL of washing solution with working concentration is obtained for later use. Adding 50 μ L of sample to be tested into each well, setting 2 wells for negative and positive control, adding 50 μ L of negative control or positive control into each well, and setting 1 well for blank control; adding 50 μ L of enzyme conjugate into each well, mixing, sealing, and incubating at 37 deg.C for 30 min; then, manually washing the plate, namely discarding liquid in the holes, filling the holes with washing liquid provided by the kit, standing for 5s, spin-drying, repeating for five times, and then drying; adding 50 mu L (or 1 drop) of color-developing agent A solution and color-developing agent B solution provided by the kit into each well after the plate washing is finished, fully and uniformly mixing, sealing a plate, incubating at 37 ℃ for 15min, then adding 50 mu L (or 1 drop) of stop solution into each well, and uniformly mixing; and finally, reading by using a microplate reader, taking the wavelength of 450nm, checking zero by using a blank hole, and then reading the OD value of each hole.
HBsAg detection refers to the hepatitis B virus surface antigen diagnostic kit provided by Shanghai Korea company. Firstly, diluting 25 times of concentrated washing solution provided in a kit into 1 time of working concentration, and the specific method comprises the following steps: measuring 480mL of purified water, adding 20mL of concentrated washing solution, and fully and uniformly mixing to obtain 500mL of washing solution with working concentration for later use; then adding 75 μ L of sample to be tested (three replicates for each sample) and negative and positive controls into the reaction well (reserving 3 negative control wells, 1 positive control well and 1 suggested blank control well altogether), covering the reaction plate with mounting paper, and incubating the reaction plate at 37 deg.C for 60 min; taking out the reaction plate, tearing off the sealing sheet, adding 50 mu L of enzyme conjugate into the hole into which the sample to be detected and the negative and positive controls are added, manually and lightly shaking for 10s, covering the reaction plate with sealing sheet paper, and incubating the reaction plate at 37 ℃ for 30 min; taking out the reaction plate, tearing off the sealing paper, and washing the reaction plate for 5 times (manual plate washing: discarding liquid in holes, filling the holes with a prepared washing solution with working concentration, standing for 30-60s, spin-drying, repeating for 5 times, and then patting dry on clean absorbent paper); immediately after washing, 50 μ L of each of the color-developing agent A and the color-developing agent B provided by the kit was added to all the wells, mixed well, and gently shaken by hand for 10 s. Then, the reaction plate was covered with a sealing plate, incubated at 37 ℃ for 30min, 50. mu.L of stop solution was added to each well, and the reaction was shaken for 5 seconds to mix well. And finally, reading by using a microplate reader, taking the wavelength of 450nm, checking zero by using a blank hole, and then reading the OD value of each hole.
The specific method for analyzing the core antigen HBcAg immunofluorescence of HBV comprises the following steps: taking 1 well of 12-well plate as an example, after at least 8 days of cell infection, the old culture solution is aspirated, washed with PBS at normal temperature for 1 time, immediately fixed with ice-cold 4% paraformaldehyde 0.5mL at normal temperature for 10-15 min; washing with PBS on a horizontal shaker for 3 times, 5min each time; to enhance binding of the antibody to intracellular proteins, 0.5mL of PBS containing 0.25% v/v Triton100 was treated at room temperature for 45 min; washing with PBS on a horizontal shaker for 3 times, 5min each time; blocking, adding a proper amount of blocking solution (PBS solution containing 5% v/v goat normal serum and 2% m/v BSA), and horizontally shaking at normal temperature for incubation for at least 1 h; diluting antibody with PBS containing 3% goat normal serum at HBcAg rabbit polyclonal antibody (purchased from Dako of Denmark) dilution ratio of 1:400, and incubating overnight at 4 deg.C in shaking table; washing with PBS solution containing 3% goat normal serum for 3 times (10 min each time) on a horizontal shaker to remove non-specifically bound antibody; the secondary antibody goat anti-rabbit lgG coupled DyLight-488 (purchased from Thermo Fisher Scientific Inc. of USA) is diluted to PBS solution containing 3% goat normal serum at a ratio of 1:400, and incubated in dark for 1h at normal temperature, after which the operation is uniformly protected from light; washing with PBS solution containing 3% goat normal serum for 2 times (10 min each) on horizontal shaker, adding 1:1000 DAPI (from Roche, Switzerland) for staining cell nucleus during the third washing, and incubating at room temperature for 10 min; washing again to remove residual DAPI for 10-20 min; an appropriate amount of PBS solution containing 3% goat normal serum was added to prevent the cells from being deformed by drying during observation. Finally, the fluorescence signal was observed using a Leica DFC425C fluorescence microscope (available from Leica, Inc., Germany).
Analysis of results
(1) Analysis of hepatitis B virus infected pig primary hepatocytes
As shown in fig. 8 and fig. 9, the secretion levels of hepatitis b virus surface antigen (abbreviated as HBsAg in english) and hepatitis b virus e antigen (abbreviated as HBeAg in english) on days 2, 4, 6 and 8 after infection of hepatitis b virus by a swine primary hepatocyte or a human hepatoma cell line Huh7D stably expressing hNTCP are shown, respectively. On the one hand, HBsAg and HBeAg show ascending trend in sequence in the stable expression pig primary hepatocytes along with the culture time, and the level of HBsAg and HBeAg is similar to that of a positive control. And a human hepatoma cell line Huh7D stably expressing hNTCP, the virus anti-raw water thereofThe level is weak positive, which is obviously lower than the primary pig liver cell which stably expresses hNTCP. Simultaneously, the two cells are added with anti-hepatitis B virus invasion small peptide Myr-preS1 during infection2-47Later, the HBsAg and HBeAg levels were down-trending or negative, indicating that hepatitis B virus infection is mediated by the receptor hNTCP. Intracellular viral core antigen (HBcAg) positive immunofluorescence signals (fig. 10-15) more fully demonstrate the susceptibility of swine primary hepatocytes stably expressing hNTCP to HBV. In summary, the above data demonstrate that porcine primary hepatocytes stably expressing hNTCP are a superior model for hepatitis b virus infection compared to the human hepatoma cell line Huh7D stably expressing hNTCP.
(2) Anti-hepatitis B virus invasion drug test based on primary pig hepatocytes
For the anti-HBV invasion inhibitor test, anti-hepatitis B virus invasion small peptide Myr-preS12-47Can be specifically combined with hNTCP so as to block the combination of hepatitis B virus and receptor hNTCP. 30min before infection, Myr-preS12-47Adding into culture medium at different concentrations (0nmol/l, 0.5nmol/l, 5nmol/l, 50nmol/l, 500nmol/l), and Myr-preS1 at infection time2-47Adding the culture medium into the infected solution at different concentrations (0nmol/l, 0.5nmol/l, 5nmol/l, 50nmol/l, 500nmol/l), incubating with cells for 16-24h, removing the infected solution, washing with Phosphate Buffered Saline (PBS) for 3 times, adding fresh maintenance culture solution PMM, and changing to 1 fresh culture solution 2 days; the old culture solution was collected for enzyme-linked immunosorbent assay, and the secretion levels of hepatitis B virus surface antigen (abbreviated as HBsAg in English) and hepatitis B virus e antigen (abbreviated as HBeAg in English) were analyzed.
The results are shown in FIG. 16, Myr-preS12-47Inhibit the secretion level of hepatitis B virus surface antigen (abbreviated as HBsAg in English) and hepatitis B virus e antigen (abbreviated as HBeAg in English) in a dose-dependent manner. Wherein 50nmol/l of small peptide can inhibit 100% of virus infection, and 0.5nmol/l of small peptide can inhibit 50% of virus antigen secretion, which is consistent with literature reports, and indicates that the hepatitis B virus infection model based on primary pig hepatocytes can be used for anti-hepatitis B virus drug efficacy tests.
Therefore, the hNTCP can be efficiently and stably expressed in the porcine primary hepatocytes, the original differentiation state of the human primary hepatocytes is simulated, the high-efficiency HBV infection and the test of HBV infection resistant drugs are supported, the hNTCP and the test method have the advantages of unlimited sources, small cell batch difference, no medical ethics and the like, and the hNTCP and the test method are expected to replace the human primary hepatocytes to play a main role in HBV basic research and HBV drug evaluation.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A method for establishing a hepatitis B virus infected cell model by utilizing primary pig hepatocytes and hNTCP recombinant lentiviruses is characterized by comprising the following steps of:
step 1: preparation of hNTCP recombinant lentivirus concentrate
Transfecting a lentivirus packaging plasmid pCMV dr8.91, a lentivirus packaging plasmid pMD.2G and an hNTCP overexpression plasmid pWPI-hNTCP-Puro into 293T cells simultaneously according to the mass ratio of 2:1:3 by using a calcium phosphate transfection method, collecting a culture supernatant containing lentiviruses 48h after transfection, and concentrating to obtain an hNTCP recombinant lentivirus concentrate;
wherein, the calcium phosphate transfection method specifically comprises the following steps: respectively adding 450 mu L of deionized water, 50 mu L of 2mol/L calcium chloride solution, 10 mu g of lentivirus packaging plasmid pCMV dr8.91, 5 mu g of lentivirus packaging plasmid pMD.2G and 15 mu g of hNTCP overexpression plasmid pWPI-hNTCP-Puro into a first centrifuge tube under the aseptic condition, and uniformly mixing to form a transfection solution I; adding 500 mu L of 2 XHBS solution into another centrifugal tube, shaking while dropping the transfection solution I to form a transfection solution II, and standing at normal temperature for 20 min; the transfection solution II was added to 293T cell culture dishes with a diameter of 10cm, a cell fusion degree of 70-80%, containing 10mL of DMEM transfection medium, at 37 ℃ with 5% CO2After the culture is carried out in the incubator for 12 hours, the DMEM transfection culture solution is changed into a fresh DMEM culture solution, namely the transfection is finished;
the concentration method comprises the following steps: putting lentivirus supernatant into a 4 ℃ horizontal centrifuge, centrifuging for 10min at 3800 Xg/min, filtering with a 0.45-micron filter membrane, adding 15mL of filtrate into a 100KD ultrafiltration tube, transferring the 100KD ultrafiltration tube into the 4 ℃ horizontal centrifuge, centrifuging for 30min at 4000 Xg/min to obtain hNTCP recombinant lentivirus concentrate;
step 2: preparation of well digested liver tissue
Digesting and separating primary pig hepatocytes by a two-step collagenase perfusion method to obtain completely digested liver tissues; wherein, the two-step collagenase perfusion method comprises the steps of perfusing fresh pig liver tissue for 20min by a perfusion solution I precooled at 4 ℃ until blood in the liver tissue is washed clean, and perfusing for 30min by a perfusion solution II preheated at 37 ℃ until the liver tissue loses elasticity;
and step 3: preparation of porcine Primary hepatocytes
Transferring the completely digested liver tissue obtained in the step 2 to a culture dish containing cell washing liquid to obtain a primary pig hepatocyte cell suspension;
and 4, step 4: preparation of Single cell suspension of porcine Primary hepatocytes
Screening the porcine primary hepatocyte cell suspension obtained in the step 3 through a cell sieve to obtain a porcine primary hepatocyte single cell suspension;
and 5: preparation of cell suspension for plating
Centrifuging the single cell suspension obtained in the step 4, re-suspending with a cell washing solution, repeating the centrifugation for 3 times, and re-suspending the cells with a plating culture solution to obtain a cell suspension for plating;
step 6: cell plating and culture
The cell suspension for plating obtained in step 5 was mixed at (1.5-2.5). times.105Viable cells/cm2Inoculating to collagen-coated culture plate or dish, adding plating culture solution, shaking, and culturing at 37 deg.C and 5% CO2After culturing for 4-6h in an incubator, replacing the plating culture solution with a hepatocyte maintenance culture medium PMM to obtain cultured primary porcine hepatocytes;
and 7: hNTCP recombinant lentivirus infection of primary hepatocytes of swine
Infecting the cultured primary porcine hepatocytes obtained in the step 6 with the hNTCP recombinant lentivirus concentrated solution obtained in the step 1 by using the infection number of 1 as an infection number, incubating for 24h, changing into a fresh hepatocyte maintenance culture medium PMM, changing for 1 time every 2 days, and detecting hNTCP protein expression on the 4 th day after infection to obtain hNTCP protein over-expressed primary porcine hepatocytes;
and 8: establishing hepatitis B virus infected cell model
And (3) adding a hepatitis B virus infection blocker into the hNTCP protein overexpressed pig primary hepatocytes obtained in the step (7), incubating for 30min, then infecting hepatitis B virus with the infection complex number of 1000, changing into a fresh hepatocyte maintenance culture medium PMM after infecting for 16-24h, changing for 1 time every 2 days, and detecting hepatitis B virus infection indexes on the 8 th day after infection to obtain a hepatitis B virus infected cell model established by using the pig primary hepatocytes and the hNTCP recombinant lentiviruses.
2. The method for establishing the hepatitis B virus infected cell model by using the porcine primary hepatocytes and the hNTCP recombinant lentivirus according to claim 1, wherein the DMEM transfection culture solution is DMEM culture medium added with 10% v/v fetal bovine serum; the fresh DMEM culture solution is prepared by adding 100units/mL penicillin, 100mg/mL streptomycin and 10% v/v fetal calf serum into a DMEM culture medium.
3. The method for establishing the hepatitis B virus infected cell model by using the porcine primary hepatocytes and the hNTCP recombinant lentiviruses as claimed in claim 1, wherein the perfusion solution I is a mixed solution containing 2mmol/l ethylene glycol bis (2-aminoethylether) tetraacetic acid in D-Hank's solution, the pH value is 7.4, the mixed solution is filtered by a 0.22-micron filter membrane and is stored at 4 ℃; the perfusion solution II is a mixed solution containing 5mmol/l calcium chloride, 0.1g/l magnesium chloride hexahydrate, 0.3g/l collagenase IV, 1.0g/l type II separating enzyme and 50mg/l nuclease in D-Hank's solution, the pH value is 7.4, the mixed solution is filtered by a 0.22 mu m filter membrane and is stored at 4 ℃.
4. The method for establishing the hepatitis B virus infected cell model by using the porcine primary hepatocytes and the hNTCP recombinant lentiviruses as claimed in claim 1, wherein in the step 5, the centrifugation conditions are 50 Xg at 4 ℃ for 5 min; the plating culture solution is prepared by adding 1% v/v ITS + premix, 2mmol/l glutamine, 10 mu g/l epidermal growth factor, 18mg/l hydrocortisone, 40 mu g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin and 5% v/v fetal calf serum into William's E culture medium.
5. The method for establishing a hepatitis B virus infected cell model using porcine primary hepatocytes and hNTCP recombinant lentivirus according to claim 1, wherein the collagen-coated culture plate or dish is prepared by the following steps in step 6: adding 172 mu L of glacial acetic acid into 100mL of deionized water, adding IV type rat tail collagen with the final concentration of 50 mu g/mL, filtering and sterilizing by using a 0.22 mu m filter membrane under the aseptic condition to obtain a collagen working solution, and keeping the collagen working solution at 4 ℃ for later use; the collagen working solution was added at a concentration of 5. mu.g/cm2Adding the coating amount into a culture plate or a culture dish to be coated, incubating at normal temperature for 1h, sucking out the collagen working solution, naturally drying, sealing, and preserving at 4 ℃; the hepatocyte maintenance medium PMM is prepared by adding 1% v/v ITS + premix, 2mmol/l glutamine, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisone, 40 μ g/l dexamethasone, 100units/mL penicillin, 100mg/mL streptomycin and 2% v/v DMSO into William's E culture medium.
6. The method for establishing the hepatitis B virus-infected cell model by using the porcine primary hepatocytes and the hNTCP recombinant lentiviruses as claimed in claim 1, wherein in step 8, the concentrations of the hepatitis B virus infection blocking agents are 500nmol/L, 50nmol/L, 5nmol/L, 0.5nmol/L, 0.05nmol/L and 0nmol/L respectively, and the hepatitis B virus infection blocking agent is Myr-PreS12-47(ii) a The hepatitis B virus infection indexes are hepatitis B virus surface antigen, hepatitis B virus e antigen and intracellular hepatitis B virus core antigen.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509751A (en) * | 2013-07-08 | 2014-01-15 | 康珞生物科技（武汉）有限公司 | Co-culturing method of human primary hepatocytes and liver nonparenchymal cells |
| CN103740637A (en) * | 2014-01-09 | 2014-04-23 | 康珞生物科技（武汉）有限公司 | Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method |
| WO2014072526A1 (en) * | 2012-11-12 | 2014-05-15 | Ruprecht-Karls-Universität Heidelberg | Development of hbv- and/or hdv-susceptible cells, cell lines and non-human animals |
-
2017
- 2017-11-29 CN CN201711220061.9A patent/CN107988260B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014072526A1 (en) * | 2012-11-12 | 2014-05-15 | Ruprecht-Karls-Universität Heidelberg | Development of hbv- and/or hdv-susceptible cells, cell lines and non-human animals |
| CN103509751A (en) * | 2013-07-08 | 2014-01-15 | 康珞生物科技（武汉）有限公司 | Co-culturing method of human primary hepatocytes and liver nonparenchymal cells |
| CN103740637A (en) * | 2014-01-09 | 2014-04-23 | 康珞生物科技（武汉）有限公司 | Method for separating, freezing and resuscitating human fetal hepatocytes susceptible to hepatitis B virus and application of method |
Non-Patent Citations (4)
| Title |
|---|
| Alexander König等.Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes.《Journal of Hepatology》.2014,第61卷 * |
| hNTCP‑expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs;Ming Zhou等;《Mol Med Rep》;20190829;第20卷(第4期);3820-3828 * |
| 应用慢病毒载体携带HBV基因组DNA制备乙型肝炎病毒感染小鼠模型的影响因素分析;王文等;《病毒学报》;20170930;第33卷(第5期);668-675 * |
| 慢病毒介导过表达NTCP的NTCP-SK-Hep1细胞系构建;勾英等;《遵义医学院学报》;20170430;第40卷(第2期);150-155 * |
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