CN107988260A - A kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus - Google Patents
A kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus Download PDFInfo
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Abstract
The invention discloses a kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus, belong to cell renovation technique field.It includes the following steps：Step 1：Prepare hNTCP recombinant slow virus concentrates；Step 2：Prepare and digest complete hepatic tissue；Step 3：Prepare pig primary hepatocyte；Step 4：Prepare pig primary hepatocyte single cell suspension；Step 5：Prepare bed board cell suspension；Step 6：Plating cells and culture；Step 7：HNTCP recombinant slow virus infection is carried out to pig primary hepatocyte；Step 8：Establish hepatitis B virus infection cell model.The present invention builds the recombinant slow virus of the gene containing hNTCP in vitro, by the slow virus of high efficiency of infection by hNTCP gene integrations into pig primary hepatocyte genome, realize that hNTCP stablizes and be overexpressed, support pig primary hepatocyte hepatitis B infected.
Description
Technical field
The present invention relates to one kind hepatitis B virus infection cell membrane is established using pig primary hepatocyte and hNTCP recombinant slow virus
The method of type, belongs to cell renovation technique field.
Background technology
Hepatitis B (english abbreviation HBV) infection is Chinese " state's disease ", and China there are about 90,000,000 Hepatitis B carriers, often
Year dies of the number about 1,000,000 of hepatitis B infected relevant disease.Due to lacking suitable HBV infection cell model, Anti-HBV drugs
Research and development relatively lag behind.Although hepatitis B vaccine can protect the health, people invades and harasses from hepatitis B, is taken for the huge hepatitis B of radix
Band person, there is no curative medicine at present.HBV infection cell model and animal model are researched and developed most important for Anti-HBV drugs.
For a long time, Anti-HBV drugs screening with evaluation cell model be confined to liver cancer cell lines such as HepG2,
HepG2.2.15, Huh7 etc..Since these cell lines are dedifferented seriously, differed compared with internal liver cell on differentiation state
It is very remote, so in screening with that cannot reflect the real antiviral effect of medicine and cytotoxicity during evaluation Anti-HBV drugs.Na+/
Ox sulphur cholate cotransports polypeptide (human Na+/ taurocholate cotransporting polypeptide, hNTCP) quilt
After being proved to be hepatitis B acceptor, HBV infection cell model is constructed based on liver cancer cell lines HepG2, Huh7, in HBV basic research
And played an important role in Anti-HBV drugs research and development.But the differentiation state of this kind of cell line is relatively low, and infectious effect is paid no attention to
Think.
Reset condition of the human primary hepatocyte due to maintaining environment in liver, " gold is acknowledged as in Anti-HBV drugs research and development
Standard ".Human primary hepatocyte is the best model of HBV researchs and antiviral drugs research and development, but there are source for human primary hepatocyte
The shortcomings of limited, batch wise differences are big, medical ethics problems, this cell can not be researched and developed in HBV basic research and Anti-HBV drugs
In be widely used.Still do not supported it is experimentally confirmed that the liver cancer cells for coming from mouse and rat are tied up to after being overexpressed hNTCP
HBV infection, illustrates height preferendum and species specificity of the HBV to human liver cell, and the liver cell of other animals does not prop up probably
HBV infection is held, this results in a kind of prejudice.
It is primary in pig based on pig is close with people's genetic distance, liver transcript profile similitude and Swine serum epidemiology survey
HBV infection is very likely supported after being overexpressed hNTCP in liver cell, so as to prepare the susceptible primary cell models of HBV and move
Thing infection model.Furthermore due to common transfection reagent such as Lipo2000 and electrotransfection, it is extremely difficult to realize to the efficient of primary hepatocyte
Genetic manipulation.And slow virus can be realized and the efficient infection of primary hepatocyte is expressed with gene integration.But at present, there has been no utilization
The method that pig primary hepatocyte and hNTCP recombinant slow virus establish hepatitis B virus infection cell model.
The content of the invention
Present invention aims to solve the deficiencies of the prior art, and provides a kind of one kind utilizes pig primary hepatocyte and hNTCP restructuring
The method that slow virus establishes hepatitis B virus infection cell model.The present invention builds the recombinant slow virus of the gene containing hNTCP in vitro,
By the slow virus of high efficiency of infection by hNTCP gene integrations into pig primary hepatocyte genome, realize that hNTCP stablized table
Reach, support pig primary hepatocyte hepatitis B infected.
The technical solution that the present invention solves above-mentioned technical problem is as follows：One kind is recombinated using pig primary hepatocyte and hNTCP
The method that slow virus establishes hepatitis B virus infection cell model, includes the following steps：
Step 1：Prepare hNTCP recombinant slow virus concentrates
Using calcium phosphate transfection method, by slow virus packaging plasmid pCMV dr8.91, slow virus packaging plasmid pMD.2G and
HNTCP is overexpressed plasmid pWPI-hNTCP-Puro in mass ratio 2:1:3, while be transfected into 293T cells, 48h is received after transfection
Collect the culture supernatant containing slow virus, after concentration, obtain hNTCP recombinant slow virus concentrates；
Step 2：Prepare and digest complete hepatic tissue
By two step collagenase perfusion methods, digestion separation pig primary hepatocyte, obtains digesting complete hepatic tissue；
Step 3：Prepare pig primary hepatocyte
The complete hepatic tissue of digestion that step 2 is obtained, is transferred in the culture dish containing cell washing lotion, it is primary to obtain pig
Hepatocyte cell suspension；
Step 4：Prepare pig primary hepatocyte single cell suspension
The pig primary hepatocyte cell suspension that step 3 is obtained, crosses cell sieve, and it is unicellular outstanding to obtain pig primary hepatocyte
Liquid；
Step 5：Prepare bed board cell suspension
The single cell suspension that step 4 is obtained centrifuges, and after being resuspended with cell washing lotion, centrifugation 3 times is repeated, with bed board culture
Cell is resuspended in liquid, obtains bed board cell suspension；
Step 6：Plating cells and culture
The bed board cell suspension that step 5 is obtained is with (1.5-2.5) × 105Living cells/cm2Inoculum density, be inoculated into
In the coated culture plate of collagen or culture dish, bed board nutrient solution is added, is shaken up, in 37 DEG C, 5%CO24-6h is cultivated in incubator
Afterwards, change bed board nutrient solution into liver cell and maintain culture medium PMM, obtain cultured pig primary hepatocyte；
Step 7：HNTCP recombinant slow virus infection is carried out to pig primary hepatocyte
The hNTCP recombinant slow virus concentrates that step 1 is obtained, using infection multiplicity as the 1 obtained culture of infection step 6 well
Pig primary hepatocyte, infection is incubated 24h, changes fresh liver cell into and maintain culture medium PMM, change 1 time within every 2 days, the 4th after infection
Its detection hNTCP protein expression, obtains the pig primary hepatocyte of hNTCP protein overexpressions；
Step 8：Establish hepatitis B virus infection cell model
In the pig primary hepatocyte of the hNTCP protein overexpressions obtained to step 7, first add hepatitis B virus infection and block
Agent, after being incubated 30min, then using infection multiplicity as 1000 infection hepatitis Bs, fresh liver cell dimension is changed into after infecting 16-24h
Culture medium PMM is held, is replaced 1 time within every 2 days, the 8th day detection hepatitis B virus infection index after infection, that is, obtain described primary using pig
Liver cell and hNTCP recombinant slow virus establish hepatitis B virus infection cell model.
The present invention is overexpressed by the hNTCP of lentivirus mediated, confirms that pig primary hepatocyte is supported through hepatitis B virus infection
Hepatitis B virus infection, its neurological susceptibility to hepatitis B surely turns liver cancer cell lines higher compared to hNTCP, and can be used for anti-hepatitis B
Virus drugs are tested.In addition, pig primary hepatocyte, relative to human primary hepatocyte, source is unrestricted, difference between cell batch
It is small, there is no medical ethics problems, be preferable cell model.
Based on the above technical solutions, the present invention can also be improved as follows.
Further, in step 1, slow virus packaging plasmid pCMV dr8.91, slow virus packaging plasmid pMD.2G and
HNTCP is overexpressed plasmid pWPI-Puro and shares Addgene websites both from global plasmid.HNTCP is overexpressed plasmid pWPI-
HNTCP-Puro is to be cloned into hNTCP in pWPI-Puro carriers by molecular cloning method.293T cells, by people's kidney epithelium
293 cell of cell line can express SV40 large T antigens, be that production slow virus is common by the derivative cell line of gene technology
Cell line, buy in China typical culture collection center (China Center for Type Culture Collection,
Abbreviation CCTCC).
Further, in step 1, the calcium phosphate transfection method is specially：Under aseptic condition, into first centrifuge tube respectively
It is sick slowly to add 450 μ L deionized waters, 50 μ L 2mol/L calcium chloride solutions, 10 μ g slow virus packaging plasmids pCMV dr8.91,5 μ g
Malicious packaging plasmid pMD.2G and 15 μ g hNTCP is overexpressed plasmid pWPI-hNTCP-Puro, and mixing forms Transfection solution I；Another
500 μ L 2 × HBS solution, side concussion are added in one centrifuge tube, side instills Transfection solution I, forms Transfection solution II, and room temperature is quiet
Put 20min；Transfection solution II is added to a diameter of 10cm, cell fusion degree 70-80%, transfects and cultivates containing 10mL DMEM
In the 293T Tissue Culture Dish of liquid, in 37 DEG C, 5%CO2After cultivating 12h in incubator, DMEM transfection nutrient solutions are changed into fresh
DMEM nutrient solutions, i.e. transfection terminate.
Further, 2 × HBS solution, is that 8.00g sodium chloride, 0.38g chlorinations are added in 400mL deionized waters
Potassium, 0.10g disodium hydrogen phosphates, 5.00g hydroxyethyl piperazineethanesulfonic acids, 1.00g glucose, it is 7.05 to adjust pH value, is settled to
500mL, it is degerming with 0.22 μm of membrane filtration, it is stored in -20 DEG C of gained.
Further, the DMEM transfections nutrient solution, is that 10%v/v hyclones are added in DMEM culture mediums.Wherein,
DMEM culture mediums, hyclone, are purchased to GIBCO companies of the U.S..
It is using above-mentioned further beneficial effect：DMEM transfects nutrient solution antibiotic-free.
Further, the fresh DMEM nutrient solutions, be added in DMEM culture mediums 100units/mL penicillin,
100mg/mL streptomysins and 10%v/v hyclones.
Wherein, DMEM culture mediums, hyclone, penicillin, streptomysin, are purchased to GIBCO companies of the U.S..
Further, in step 1, the method for the concentration is：First slow virus supernatant is placed in 4 DEG C of horizontal centrifuges,
3800 × g/min, centrifuges 10min, after 0.45 μm of membrane filtration, takes 15mL filtered fluids to be added in 100KD super filter tubes, then
Above-mentioned 100KD super filter tubes are transferred in 4 DEG C of horizontal centrifuges again, 4000 × g/min, after centrifuging 30min, that is, obtain hNTCP
Recombinant slow virus concentrate.
It is using above-mentioned further beneficial effect：Concentration is divided into be centrifuged twice, is centrifuged for the first time, is to tentatively remove
Dead cell or big cell fragment.0.45 μm of membrane filtration, is to remove small cell fragment.Second centrifuges, be in order to
Molecular cut off is more than the molecule of 100 dalton, including hNTCP recombinant slow virus, to achieve the purpose that concentration.
Above-mentioned 100KD super filter tubes, purchased from Millipore companies of the U.S..
Further, in step 2, the two steps collagenase perfusion method, is that fresh pig liver is first passed through 4 DEG C of precoolings
Primer solution I irrigates 20min, until the blood in hepatic tissue is rinsed totally, then is irrigated with the primer solution II of 37 DEG C of preheatings
30min, until hepatic tissue follows the string.
Further, the primer solution I is double (2- amino ethyl ethers) containing 2mmol/l ethylene glycol in D-Hank ' s solution
The mixed liquor of tetraacethyl, 7.4,0.22 μm of membrane filtration of pH value, 4 DEG C of preservations.
Further, the primer solution II is containing 5mmol/l calcium chloride, six water chlorine of 0.1g/l in D-Hank ' s solution
Change magnesium, 0.3g/l type Ⅳ collagenases, 1.0g/l II types separation enzyme, the mixed liquor of 50mg/l nucleases, 7.4,0.22 μm of pH value
Membrane filtration, 4 DEG C of preservations.
Wherein, type Ⅳ collagenase, purchased from Sigma Co., USA.II types separate enzyme, i.e. Dispase II, purchased from Switzerland
Roche companies.Nuclease, i.e. DNase I, purchased from Chinese Solarbio companies.
Further, 8.00g/l sodium chloride, 0.40g/l potassium chloride, 0.06g/l bis- are contained in D-Hank ' the s solution
Water disodium hydrogen phosphate, 0.06g/l potassium dihydrogen phosphates and 0.35g/l sodium acid carbonates.
Further, in step 3, the cell washing lotion, be added in DMEM culture mediums 100units/mL penicillin,
100mg/mL streptomysins and 10%v/v hyclones.
Further, in step 4, the aperture of the cell sieve is 70 μm.
Further, in step 5, the condition of the centrifugation is 50 × g, and 4 DEG C, the time of centrifugation is 5min.
Further, in step 5, the bed board nutrient solution, is the ITS+ that 1%v/v is added in William ' s E culture mediums
Premix, 2mmol/l glutamine, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisones, 40 μ g/l dexamethasone,
100units/mL penicillin, 100mg/mL streptomysins, 5%v/v hyclones.
Wherein, William ' s E culture mediums, abbreviation WE, purchased from GIBCO companies of the U.S..ITS+premix, purchased from the U.S.
GIBCO companies.Epidermal growth factor, purchased from R＆D companies of the U.S..Hyclone, purchased from GIBCO companies of the U.S..
Further, the ITS+premix is 100 times of mother liquors, turns iron containing 6.25 μ g/mL insulin, 6.25 μ g/mL
Albumen, 6.25ng/mL selenous acid, 1.25mg/mL bovine serum albumin(BSA)s and 5.35 μ g/mL linoleic acid.
Further, in step 6, the preparation method of the coated culture plate of the collagen or culture dish is：By 172 μ L glacial acetic acid
It is added in 100mL deionized waters, adds the IV type Collagen type-I of final concentration of 50 μ g/mL, under aseptic condition, with 0.22 μm
After membrane filtration is degerming, collagen working solution is obtained, 4 DEG C of holdings are stand-by；By the collagen working solution with 5 μ g/cm2Package amount add
Into the coated culture plate of needs or culture dish, after room temperature is incubated 1h, collagen working solution is suctioned out, is sealed after naturally dry, 4 DEG C
Preservation.
Wherein, the coated culture plate of above-mentioned collagen or culture dish are before use, need to be remaining to remove with PBS cleaning once
Glacial acetic acid.
Further, in step 6, the liver cell maintains culture medium PMM, is to add 1% in William ' s E culture mediums
ITS+premix, 2mmol/l glutamine of v/v, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisones, 40 μ g/l
Sai meter Song, 100units/mL penicillin, 100mg/mL streptomysins and 2%v/v DMSO.
Wherein, William ' s E culture mediums, abbreviation WE, purchased from GIBCO companies of the U.S..ITS+premix, purchased from the U.S.
GIBCO companies.Epidermal growth factor, purchased from R＆D companies of the U.S..Hyclone, purchased from GIBCO companies of the U.S..
Further, the ITS+premix is 100 times of mother liquors, turns iron containing 6.25 μ g/mL insulin, 6.25 μ g/mL
Albumen, 6.25ng/mL selenous acid, 1.25mg/mL bovine serum albumin(BSA)s and 5.35 μ g/mL linoleic acid.
Further, in step 8, the concentration of the hepatitis B virus infection blocking agent be respectively 500nmol/L, 50nmol/L,
5nmol/L, 0.5nmol/L, 0.05nmol/L, 0nmol/L, the hepatitis B virus infection blocking agent are Myr-PreS12-47。
Above-mentioned hepatitis B virus infection blocking agent Myr-PreS12-47Purchased from gill biochemistry (Shanghai) Co., Ltd., amino acid
Sequence is Myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKV G, and wherein aminoterminal is
Myristoylation (Myristoyl) is modified.
Further, in step 8, the hepatitis B virus infection index is hepatitis B virus surface antigen, hepatitis B e in supernatant
Antigen and intracellular hepatitis B virus core antigen.
Beneficial effects of the present invention：
(1) slow virus that the present invention is built can efficiently infect pig primary hepatocyte, and hNTCP is incorporated into
In genome, the high efficiency stable expression of hNTCP is realized, simulate the reset condition of human primary hepatocyte.
(2) present invention is separated to high vigor pig primary hepatocyte, and the bed board in a manner of high density, culture medium is maintained in liver cell
The monolayer cell culture of 100% degrees of fusion is formed in PMM, hepatocyte differentiation characteristic can be maintained about 1 month, be slow-virus infection,
The time that hNTCP expression, hepatitis B virus infection and subsequent viral duplication provide abundance with release ensures.
(3) present invention establishes the pork liver primary cell that hNTCP stablizes expression, supports hepatitis B efficiently to infect, its is easy
The liver cancer cell lines that perception stablizes expression relative to hNTCP are more efficient, differentiation state higher, are expected to substitute human primary hepatocyte
And play main function in hepatitis B basic research and anti-hepatic-B virus medicine evaluation.
(4) relative to human primary hepatocyte, the pork liver primary cell that the hNTCP that the present invention obtains stablizes expression has source
It is unrestricted, cell batch difference is small, there is no the advantages that Medical Ethics；Tentatively establish novel anti-hepatitis virus and infect medicine
Thing screens the In vitro cell model with evaluation, and possibility is provided to develop the animal infection modal with intact immune system, right
Antiviral drugs research has important value.
Brief description of the drawings
Fig. 1 is the recombinant slow virus plasmid figure spectrogram that the present invention includes hNTCP genes.
Fig. 2 is the pig primary hepatocyte bed board cellular morphology figure of the 2nd day that the present invention cultivates.
Fig. 3 is the pig primary hepatocyte bed board cellular morphology figure of the 30th day that the present invention cultivates.
Fig. 4 is the Immunofluorescence test figure compareed that hNTCP of the present invention expresses the Bel7402 Huh7D with positioning.
Fig. 5 is the Immunofluorescence test figure for the hNTCP that hNTCP of the present invention expresses the Bel7402 Huh7D with positioning.
Fig. 6 is the Immunofluorescence test figure compareed that hNTCP of the present invention expresses the pig primary hepatocyte with positioning.
Fig. 7 is the Immunofluorescence test figure for the hNTCP that hNTCP of the present invention expresses the pig primary hepatocyte with positioning.
Fig. 8 is Hepatitis B virus e antigen of the present invention secretion detection figure.
Fig. 9 is hepatitis B virus s antigen of the present invention secretion detection figure.
The nuclear targeting detection figure that Figure 10 is hepatitis B virus infection Bel7402 Huh7D of the present invention.
Figure 11 is the core antigen immunofluorescence dye of the B-type hepatitis of hepatitis B virus infection Bel7402 Huh7D of the present invention
Chromatic graph.
Figure 12 is that the nuclear targeting of hepatitis B virus infection Bel7402 Huh7D of the present invention and the core of B-type hepatitis resist
Former immunofluorescence dyeing overlay chart.
Figure 13 is the nuclear targeting detection figure of hepatitis B virus infection pig primary hepatocyte of the present invention.
Figure 14 is the core antigen immunofluorescence dyeing figure of the B-type hepatitis of hepatitis B virus infection pig primary hepatocyte of the present invention.
Figure 15 is that the nuclear targeting of hepatitis B virus infection pig primary hepatocyte of the present invention and the core antigen of B-type hepatitis are exempted from
Epidemic disease fluorescent staining overlay chart.
Figure 16 is the test and evaluation figure that anti-hepatitis virus of the present invention invades inhibitor.
Embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Embodiment
The present embodiment establishes hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus
Method, includes the following steps：
Step 1：Prepare hNTCP recombinant slow virus concentrates
Using calcium phosphate transfection method, by slow virus packaging plasmid pCMV dr8.91, slow virus packaging plasmid pMD.2G and
HNTCP is overexpressed plasmid pWPI-hNTCP-Puro in mass ratio 2:1:3, while be transfected into 293T cells, 48h is received after transfection
Collect the culture supernatant containing slow virus, after concentration, obtain hNTCP recombinant slow virus concentrates.
(1) hNTCP expression plasmids are built
Slow virus packaging plasmid pCMV dr8.91, slow virus packaging plasmid pMD.2G and hNTCP are overexpressed plasmid pWPI-
Puro shares Addgene websites (http both from global plasmid://www.addgene.org/).Obtained from human liver tissue
People Na+/ ox sulphur cholate cotransports the gene coded sequence of polypeptide (hNTCP), will with the molecular cloning method linked by digestion
HNTCP is cloned into pWPI-Puro empty carriers, obtains hNTCP slow virus expression plasmid pWPI-hNTCP-Puro (Fig. 1).293T
Cell, by 293 cell of people's renal epithelial cell system by the derivative cell line of gene technology, can express SV40 large T antigens, be raw
The common cell line of slow virus is produced, is bought in China typical culture collection center (China Center for Type
Culture Collection, abbreviation CCTCC).
(2) lentivirus production
Recovery 293T cells, 37 DEG C, 5%CO are incubated at using fresh DMEM nutrient solutions2In incubator, 24h is cultivated.Utilize
Trypsin digestion cell is into unicellular, with 1:3 passage bed boards transfect nutrient solution into the Tissue Culture Dish of a diameter of 10cm in DMEM
In continue to cultivate 12h, 293T cell fusions degree about 70-80%, by calcium phosphate transfection method by plasmid transfection into cell.
Fresh DMEM nutrient solutions, are that DMEM culture mediums (being purchased from GIBCO companies of the U.S.) add (purchase of 10%v/v hyclones
From GIBCO companies of the U.S.), 100units/mL penicillin and 100mg/mL streptomysins.DMEM transfects nutrient solution, is cultivated for DMEM
10%v/v hyclones (being purchased from GIBCO companies of the U.S.) are added in base (being purchased from GIBCO companies of the U.S.).
Calcium phosphate transfection method is specially：Under aseptic condition, be separately added into first centrifuge tube 450 μ L deionized waters,
50 μ L2mol/L calcium chloride solutions, 10 μ g slow virus packaging plasmids pCMV dr8.91,5 μ g slow virus packaging plasmid pMD.2G and
15 μ g hNTCP are overexpressed plasmid pWPI-hNTCP-Puro, and mixing forms Transfection solution I；500 are added in another centrifuge tube
μ L2 × HBS solution, side concussion, side instill Transfection solution I, form Transfection solution II, and room temperature stands 20min；By Transfection solution II
It is added in a diameter of 10cm, cell fusion degree 70-80%, the 293T Tissue Culture Dish containing 10mL DMEM transfection nutrient solutions,
In 37 DEG C, 5%CO2After cultivating 12h in incubator, DMEM transfection nutrient solutions are changed into fresh DMEM nutrient solutions, i.e. transfection terminates.
2 × HBS solution, is that 8.00g sodium chloride, 0.38g potassium chloride, 0.10g phosphoric acid hydrogen are added in 400mL deionized waters
Disodium, 5.00g hydroxyethyl piperazineethanesulfonic acids, 1.00g glucose, it is 7.05 to adjust pH value, is settled to 500mL, with 0.22 μm of filter
Membrane filtration is degerming, is stored in -20 DEG C of gained.
DMEM transfects nutrient solution, is that 10%v/v hyclones are added in DMEM culture mediums (being purchased from GIBCO companies of the U.S.)
(being purchased from GIBCO companies of the U.S.).
Fresh DMEM nutrient solutions, are that 100units/mL penicillin is added in DMEM culture mediums (being purchased from GIBCO companies of the U.S.)
(being purchased from GIBCO companies of the U.S.), 100mg/mL streptomysins (being purchased from GIBCO companies of the U.S.) and 10%v/v hyclones are (purchased from U.S.
GIBCO companies of state).
The method of concentration is：First slow virus supernatant is placed in 4 DEG C of horizontal centrifuges, 3800 × g/min, centrifuges 10min,
After 0.45 μm of membrane filtration, 15mL filtered fluids are taken to be added in 100KD super filter tubes (being purchased from Millipore companies of the U.S.), so
Above-mentioned 100KD super filter tubes are transferred in 4 DEG C of horizontal centrifuges again afterwards, 4000 × g/min, after centrifuging 30min, 100KD is surpassed
The viral concentration liquid (about 250 μ L) retained in chimney filter takes out, and is stored in -80 DEG C, that is, obtains hNTCP recombinant slow virus concentrates.
(3) slow virus titer determination
Before measure 1 day by liver cancer cell lines Huh7 cells with 5 × 104The density in/hole is inoculated in 96 orifice plates；Using containing 6 μ
The DMEM nutrient solutions of g/ml hexadimethrine bromides carry out 10 times of gradient dilutions to hNTCP recombinant slow virus concentrate, respectively obtain
Extension rate is 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Dilution；Suctioned out from 96 orifice plates original
DMEM nutrient solutions, above-mentioned diluted virus infection liquid is separately added into each hole, is placed in 37 DEG C, 5%CO2Cultivated in incubator
After night, the fresh DMEM nutrient solutions of 100 μ L are changed into, continue after cultivating 48h, each cell hole infected in 96 orifice plates utilizes pancreatin
Digestion is with 1:2 are passaged in 2 new culture holes, wherein 1 hole, which adds, contains 5 μ g/mL puromycins, the blank control of another 1 hole；Train again
48h is supported, calculates puromycin processing hole and cell number in blank control wells in each thinner ratio respectively by cell count, and
Calculate viral efficiency of infection and virus titer.
Wherein, viral efficiency of infection=(cell number in puromycin processing hole cell number/blank control wells) × 100%,
Viral efficiency of infection is calculated, virus titer, virus titer are calculated by the correspondence cell hole of about 10% efficiency of infection
(titer/mL)=virus efficiency of infection × without puromycin processing hole cell number × extension rate.
Step 2：Prepare and digest complete hepatic tissue
By two step collagenase perfusion methods, digestion separation pig primary hepatocyte, obtains digesting complete hepatic tissue.
Wherein, the two step collagenase perfusion method first step：The blood vessel that fresh pig liver is exposed through is irrigated molten with 4 DEG C of precoolings
Liquid I irrigates 20min, until blood is rinsed totally in hepatic tissue.Wherein, primer solution I is to contain in D-Hank ' s solution
The mixed liquor of double (2- amino ethyl ethers) tetraacethyls of 2mmol/l ethylene glycol, 7.4,0.22 μm of membrane filtration of pH value, 4 DEG C of preservations.D-
Contain 8.00g/l sodium chloride, 0.40g/l potassium chloride, 0.06g/l phosphate dihydrates disodium hydrogen, 0.06g/l phosphoric acid in Hank ' s solution
Potassium dihydrogen and 0.35g/l sodium acid carbonates
Two step collagenase perfusion method second steps：30min is irrigated with the primer solution II of 37 DEG C of preheatings, until pig liver loses
Go elasticity.Wherein, primer solution II be D-Hank ' s solution in containing 5mmol/l calcium chloride, 0.1g/l magnesium chloride hexahydrates,
0.3g/l type Ⅳ collagenases (being purchased from Sigma Co., USA), 1.0g/l II types separation enzyme (Dispase II, purchased from Switzerland
Roche companies), the mixed liquor of 50mg/l nucleases (DNase I, purchased from Chinese Solarbio companies), 7.4,0.22 μ of pH value
M membrane filtrations, 4 DEG C of preservations.
Step 3：Prepare pig primary hepatocyte
The complete hepatic tissue of digestion that step 2 is obtained, is transferred in the culture dish containing cell washing lotion, carefully shakes off to disappear
The cell changed, obtains pig primary hepatocyte cell suspension.
Wherein, cell washing lotion, be addition 100units/mL penicillin in DMEM culture mediums (be purchased from GIBCO companies of the U.S.),
100mg/mL streptomysins and 10%v/v hyclones (being purchased from GIBCO companies of the U.S.).
Step 4：Prepare pig primary hepatocyte single cell suspension
The pig primary hepatocyte cell suspension that step 3 is obtained, crosses the cell sieve that aperture is 70 μm, it is thin to obtain the primary liver of pig
Born of the same parents' single cell suspension.
Step 5：Prepare bed board cell suspension
The single cell suspension that step 4 is obtained centrifuges, and centrifugal condition is：50 × g, 4 DEG C, 5min.It is resuspended with cell washing lotion
Afterwards, centrifugation 3 times is repeated, cell is resuspended with bed board nutrient solution, obtains bed board cell suspension.
Wherein, bed board nutrient solution, is added William ' s E culture mediums (abbreviation WE, purchased from GIBCO companies of the U.S.) are inner
The ITS+premix (being purchased from GIBCO companies of the U.S.) of 1%v/v, 2mmol/l glutamine, 10 μ g/l epidermal growth factor (are purchased from
R＆D companies of the U.S.), 18mg/l hydrocortisones, 40 μ g/l dexamethasone, 100units/mL penicillin, 100mg/mL strepto-s
Element, 5%v/v hyclones (being purchased from GIBCO companies of the U.S.).
ITS+premix is 100 times of mother liquors, contains 6.25 μ g/mL insulin, 6.25 μ g/mL transferrins, 6.25ng/mL
Selenous acid, 1.25mg/mL bovine serum albumin(BSA)s and 5.35 μ g/mL linoleic acid.
The cell density and cell viability of bed board cell suspension are calculated, i.e., by bed board cell suspension bed board and 0.4%
M/v platform phenol indigo plant dyeing liquors are with volume ratio 1:After 1 mixes, mixed liquor is obtained, takes 20 μ L mixed liquors to be added to tally rapidly and (is purchased from
Counterstar companies of the U.S.) in, pass through calculating instrument reading cell density and the numerical value of cell viability.
Wherein, the preparation method of 0.4%m/v platforms phenol indigo plant dyeing liquor is：0.4g platform phenol indigo plant solids are completely dissolved in 100mL
Phosphate buffered solutions in, pH value 7.2-7.4, high pressure steam sterilization, 4 DEG C holding.Phosphate buffered solutions, english abbreviation
For PBS, contain 8.0g/l sodium chloride, 0.2g/l potassium chloride, 3.58g/l disodium hydrogen phosphates and 0.24g/l biphosphates
Potassium.
Step 6：Plating cells and culture
The bed board cell suspension that step 5 is obtained is with (1.5-2.5) × 105Living cells/cm2Inoculum density, be inoculated into
In the coated culture plate of collagen or culture dish, bed board nutrient solution is added, is shaken up, in 37 DEG C, 5%CO24-6h is cultivated in incubator,
After fully allowing its adherent, change bed board nutrient solution into liver cell and maintain culture medium PMM, cultivation cycle is 30 days, is being spread respectively
The 2nd day after plate, cellular morphology was recorded by phase contrast microscope in 30 days, obtain cultured pig primary hepatocyte.
Wherein, the preparation method of the coated culture plate of collagen or culture dish is：172 μ L glacial acetic acid are added to 100mL
In ionized water, the IV type Collagen type-I of final concentration of 50 μ g/mL is added, it is degerming with 0.22 μm of membrane filtration under aseptic condition
Afterwards, collagen working solution is obtained, 4 DEG C of holdings are stand-by；By the collagen working solution with 5 μ g/cm2Package amount be added to need it is coated
In culture plate or culture dish, after room temperature is incubated 1h, collagen working solution is suctioned out, is sealed after naturally dry, 4 DEG C of preservations.Above-mentioned glue
The culture plate or culture dish of primordial covering are before use, need to be with PBS cleaning once, to remove remaining glacial acetic acid.
Liver cell maintain culture medium PMM, be in William ' s E culture mediums add 1%v/v ITS+premix,
2mmol/l glutamine, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisones, 40 μ g/l dexamethasone, 100units/
ML penicillin, 100mg/mL streptomysins and 2%v/v DMSO.
Step 7：HNTCP recombinant slow virus infection is carried out to pig primary hepatocyte
The hNTCP recombinant slow virus concentrates that step 1 is obtained, are cultivated the 2nd day by 1 infection step 6 of infection multiplicity
Pig primary hepatocyte, infection are incubated 24h, change fresh liver cell into and maintain culture medium PMM, change 1 time within every 2 days, the 4th after infection
My god, a part of cell detects hNTCP protein expressions with the method for immunofluorescence after fixing and positions, and other cells carry out B-type hepatitis
Poison infection test, obtains the pig primary hepatocyte of hNTCP protein overexpressions.
Step 8：Establish hepatitis B virus infection cell model
In the pig primary hepatocyte of the hNTCP protein overexpressions obtained to step 7, it is respectively 500nmol/ first to add concentration
L, the hepatitis B virus infection blocking agent Myr-PreS1 of 50nmol/L, 5nmol/L, 0.5nmol/L, 0.05nmol/L, 0nmol/L2 -47(being purchased from gill biochemistry (Shanghai) Co., Ltd.), after being incubated 30min, ties up in the liver cell of the PEG 8000 containing 4%w/v
Hold and change fresh liver cell maintenance training into after 16-24h using infection multiplicity as 1000 infection hepatitis Bs, is infected in culture medium PMM
Base PMM is supported, is replaced 1 time within every 2 days, the 8th day detection hepatitis B virus infection index hepatitis B virus surface antigen (english abbreviation after infection
For HBsAg), Hepatitis B virus e antigen (english abbreviation HBeAg) and intracellular hepatitis B virus core antigen, that is, obtain the profit
Hepatitis B virus infection cell model is established with pig primary hepatocyte and hNTCP recombinant slow virus.
Detection and analysis
1st, to separated pig primary hepatocyte form and hNTCP detection of expression
On the one hand, with phase contrast microscope morphological observation different incubation time points pig primary hepatocyte form；It is another
Aspect, utilizes the expression and positioning of the method identification hNTCP of immunofluorescence.
The specific method of hNTCP immunofluorescences is：By taking 1 hole of 12 orifice plates as an example, when cell culture was to 2 months, suck old
Nutrient solution, 1 time is washed with room temperature PBS, and with ice-cold 4% paraformaldehyde 1mL, (specific formula is immediately：4g paraformaldehydes are weighed, are put
In conical flask, 80mL deionized waters are added, are put into 37 DEG C of constant water bath box, rock mixing every 1-2h, it is more after 16-24h
Polyformaldehyde can be completely dissolved, supplement deionized water to 100mL, adjust pH value to 7.2), room temperature fixes 10-15min；Existed with PBS
Washed on horizontal shaker 3 times, each 5min；For the combination of enhancing antibody and intracellular protein, contain 0.25%v/v using 0.5mL
The PBS solution room temperature processing 45min of Triton100；Washed with PBS on horizontal shaker 3 times, each 5min；Closing, adds appropriate
Confining liquid (contains 5%v/v goat normal serums, the PBS solution of 2%m/v BSA), and room temperature horizontal shaker is incubated at least 1h；Profit
Antibody, more anti-(the being purchased from Sigma Co., USA) dilution of anti-hNTCP rabbits are diluted with the PBS solution of the normal serum of goat containing 3%v/v
Ratio is 1:500,4 DEG C of shaking tables are incubated overnight；Washed again with the PBS solution containing 3% goat normal serum on horizontal shaker 3 times,
Each 10min, to remove the antibody of non-specific binding；Secondary antibody is coupled DyLight-488 for goat antirabbit lgG and (is purchased from the U.S.
Thermo Fisher Scientific Inc companies), with 1:1000 are diluted to the PBS solution containing 3% goat normal serum, in
Dark place lucifuge room temperature is incubated 1h, hereafter operates same lucifuge；Again with the PBS solution containing 3% goat normal serum in horizontal shaker
On wash 2 times, each 10min, third time clean when add 1:1000 DAPI (being purchased from Roche companies of Switzerland) is used for nuclei dyeing
Color, room temperature are incubated 10min；Clean again once to wash remaining DAPI, 10-20min；Add normal containing 3% goat in right amount
The PBS solution of serum prevents that cell is because of drying distortions in observation.Finally, (it is purchased from using Leica DFC425C fluorescence microscopes
German Leica companies) observation fluorescence signal.
Interpretation of result
(1) pig primary hepatocyte is analyzed in the morphological observation of different incubation time points
Using the isolated pig primary hepatocyte of above-mentioned two steps collagenase perfusion method under condition of culture of the present invention
Show obvious differentiated hepatocellular form.After bed board the 2nd day (Fig. 2), pig primary hepatocyte is in polygon, and nucleus is round and high
It is bright, organelles；After bed board the 30th day (Fig. 3), liver cell state is fair, and slight aging, cell fragment occurs in pig cell
Relative to the 2nd day showed increased.Since the incubation times of 30 days can meet slow-virus infection, hepatitis B virus infection and medicine
The required time cycles such as test, present inventor are determined as the time point at the terminal of culture.Picture scale is 150 μ
m。
(2) Immunofluorescence test of hNTCP expression and positioning
Using Bel7402 Huh7D as control, table of the acceptor hNTCP albumen in pig cell is confirmed by immunofluorescence
Reach and position.As Figure 4-Figure 7, in Bel7402 Huh7D, the cell for only infecting slow virus just expresses hNTCP
Albumen, and hNTCP albumen is primarily located within cell membrane.Equally, the pig primary hepatocyte for only infecting slow virus is just expressed
HNTCP albumen, and hNTCP albumen is primarily located within cell membrane.Illustrate that hNTCP albumen can express in pig primary hepatocyte,
And positioning is correct, lay a good foundation for hNTCP protein exhibits biological functions.Picture scale is 150 μm.
2nd, the pig primary hepatocyte of HBV infection expression hNTCP and Anti-HBV activity invasion drug test
ELISA and the metainfective key index detection method of Immunofluorescence test virus
The hepatitis B virus e antigen diagnostic kit specification that HBeAg detections are provided with reference to Shanghai Xiamen Kehua.It is first
First, the cleaning solution of provided in kit 25 times of concentrations is diluted to 1 times of working concentration, specific method is：It is pure to measure 480mL
Change water, add 20mL concentrated cleaning solutions, it is stand-by into 500mL working concentration cleaning solutions after fully mixing.Added per hole and treat test sample
50 μ L of product, each sample set three repetitions, if positive and negative compare each 2 hole, negative control or each 50 μ of positive control are added per hole
L, and set 1 hole of blank control；Then 50 μ L of enzyme conjugates are added per hole, are fully mixed, sealing plate, are placed in 37 DEG C of incubation 30min；So
Afterwards, manual board-washing, that is, discard liquid in hole, and the cleaning solution provided with kit fills each hole, stands 5s, drying, repeats five times
After pat dry；Color developing agent A liquid, each 50 μ L of color developing agent B liquid (or 1 drop) that kit provides are added per hole after completing board-washing, it is fully mixed
Even, sealing plate, puts 37 DEG C of incubation 15min, adds 50 μ L of terminate liquid (or 1 drop) per hole afterwards, mixes；Microplate reader reading is finally used,
Wavelength 450nm is taken, first with blank well school zero, then reads each hole OD values.
The hepatitis b virus s antigen diagnostic kit specification that HBsAg detections are provided with reference to Shanghai Xiamen Kehua.
The cleaning solution of provided in kit 25 times of concentrations is diluted to 1 times of working concentration first, specific method is：Measure 480mL
Purified water, adds 20mL concentrated cleaning solutions, stand-by into 500mL working concentration cleaning solutions after fully mixing；Then 75 μ L are added
Sample to be tested (each three repetitions of sample) and positive and negative (reserve 3 hole of negative control, positive control 1 altogether in comparison with reacting hole
Hole, suggest reserved 1 hole of blank control), after covering reaction plate with mounting paper, reaction plate is placed in 37 DEG C of incubation 60min；Take out anti-
Plate is answered, tears mounting off, 50 μ L enzyme conjugates are added in sample to be tested and negative, positive control hole has been added, by hand gently
10s is shaken, after covering reaction plate with mounting paper afterwards, reaction plate is placed in 37 DEG C of incubation 30min；Reaction plate is taken out, tears envelope off
Piece paper, 5 (manual board-washings of washing reaction plate：Liquid in hole is discarded, each hole is filled with the working concentration cleaning solution of preparation, is stood
30-60s, drying, after being repeated 5 times, pats dry on clean blotting paper)；After washing reagent is added in all holes immediately
Each 50 μ L of color developing agent A, color developing agent B that box provides, mix, and gently shake 10s by hand.Then, will after covering reaction plate with mounting
Reaction plate puts 37 DEG C of incubation 30min, adds 50 μ L terminate liquids per hole afterwards, concussion reaction 5s, is allowed to fully mix.Finally use enzyme
Instrument reading is marked, wavelength 450nm is taken, then first then reads each hole OD values with blank well school zero.
Analyzing the specific method of the core antigen HBcAg immunofluorescences of HBV is：By taking 1 hole of 12 orifice plates as an example, cell infection
After at least 8 days, old nutrient solution is sucked, is washed 1 time with room temperature PBS, is fixed immediately with 4% ice-cold paraformaldehyde 0.5mL, room temperature
10-15min；Washed with PBS on horizontal shaker 3 times, each 5min；For the combination of enhancing antibody and intracellular protein, use
The PBS solution room temperature processing 45min of 0.5mL Triton100 containing 0.25%v/v；Wash 3 times on horizontal shaker with PBS, every time
5min；Closing, adds appropriate confining liquid (containing 5%v/v goat normal serums, the PBS solution of 2%m/v BSA), and room temperature is horizontal
Shaking table is incubated at least 1h；Antibody is diluted using the PBS solution containing 3% goat normal serum, HBcAg rabbits are more anti-(to be purchased from Denmark
Dako companies) dilution ratio is incubated overnight for 1 ︰, 400,4 DEG C of shaking tables；Again with the PBS solution containing 3% goat normal serum in level
Washed on shaking table 3 times, each 10min, to remove the antibody of non-specific binding；Secondary antibody goat antirabbit lgG is coupled DyLight-488
(purchasing to Thermo Fisher Scientific Inc companies of the U.S.) is with 1:400 are diluted to the PBS containing 3% goat normal serum
Solution, is incubated 1h in dark place lucifuge room temperature, hereafter operates same lucifuge；Again with the PBS solution containing 3% goat normal serum in water
Washed on yawing bed 2 times, each 10min, third time adds 1 when cleaning:1000 DAPI (being purchased from Roche companies of Switzerland) is used for thin
Karyon dyes, and room temperature is incubated 10min；Clean again once to wash remaining DAPI, 10-20min；Addition contains 3% mountain in right amount
The PBS solution of sheep normal serum prevents that cell is because of drying distortions in observation.Finally, Leica DFC425C fluorescence microscopies are utilized
Mirror (being purchased from Leica companies of Germany) observes fluorescence signal.
Interpretation of result
(1) hepatitis B virus infection pig primary hepatocyte is analyzed
As shown in Figure 8, Figure 9, represent to stablize the pig primary hepatocyte or Bel7402 Huh7D for expressing hNTCP respectively
2nd, 4,6,8 day hepatitis B virus surface antigen (english abbreviation HBsAg), Hepatitis B virus e antigen (English after infection hepatitis B
Be abbreviated as HBeAg) secretion level.On the one hand, HBsAg and HBeAg stablize express pig primary hepatocyte in incubation time
In ascendant trend successively, it is horizontal close with positive control for passage.And stablize the Bel7402 Huh7D of expression hNTCP, its
Viral antigen level is in weakly positive, hence it is evident that less than the pig primary hepatocyte for stablizing expression hNTCP.Meanwhile two kinds of cells are infecting
When add anti-hepatitis virus invasion small peptide Myr-preS12-47Afterwards, horizontal on a declining curve or negative, the explanations of HBsAg and HBeAg
Hepatitis B virus infection is acceptor hNTCP mediations.Intracellular virus core antigen (HBcAg) positive immunofluorescence signal (Figure 10-
The pig primary hepatocyte for Figure 15) having more fullyd illustrate stable expression hNTCP has neurological susceptibility to HBV.In short, data above explanation
Stablize the pig primary hepatocyte of expression hNTCP compared to the Bel7402 Huh7D for stablizing expression hNTCP, be more excellent
Hepatitis B virus infection model.
(2) the anti-hepatitis virus invasion drug test based on pig primary hepatocyte
For Anti-HBV activity invasion inhibitor test, anti-hepatitis virus invasion small peptide Myr-preS12-47It can occur with hNTCP special
The opposite sex combines, so that the combination of inhibition of hepatitis b virus and acceptor hNTCP.30min before infection, Myr-preS12-47With various concentrations
(0nmol/l, 0.5nmol/l, 5nmol/l, 50nmol/l, 500nmol/l) is added in nutrient solution in advance, Myr- during infection
preS12-47Equally sense is added to various concentrations (0nmol/l, 0.5nmol/l, 5nmol/l, 50nmol/l, 500nmol/l)
In dye liquor, with cell incubation 16-24h, remove infection liquid, cleaned 3 times with phosphate buffer (PBS), add fresh maintenance culture
Liquid PMM, changes 1 fresh nutrient solution for 2 days；Collect old nutrient solution and be used as enzyme-linked immunosorbent assay, analyze Hepatitis B Surface
The secretion level of antigen (english abbreviation HBsAg), Hepatitis B virus e antigen (english abbreviation HBeAg).
As a result as shown in figure 16, Myr-preS12-47Suppress hepatitis B virus surface antigen (English in a manner of dose-dependent
Be abbreviated as HBsAg), the secretion level of Hepatitis B virus e antigen (english abbreviation HBeAg).The wherein small peptide of 50nmol/l can
100% suppresses virus infection, and the small peptide of 0.5nmol/l can inhibit 50% viral antigen secretion, this is consistent with document report, says
The bright hepatitis B virus infection model based on pig primary hepatocyte can be used for anti-hepatic-B virus medicine efficacy testing.
It can be seen from the above that the present invention realizes hNTCP high efficiency stable expressions in pig primary hepatocyte, the simulation primary liver of people is thin
The original differentiation state of born of the same parents, support HBV efficiently infection and anti HBV infecting medicine test, have source do not receive limitation, carefully
Born of the same parents' batch wise differences are small, there is no the advantages that Medical Ethics, be expected to substitute human primary hepatocyte and in HBV basic research and Anti-HBV activity
Main function is played in drug evaluation.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of method for establishing hepatitis B virus infection cell model using pig primary hepatocyte and hNTCP recombinant slow virus, its
It is characterized in that, includes the following steps：
Step 1：Prepare hNTCP recombinant slow virus concentrates
Using calcium phosphate transfection method, by slow virus packaging plasmid pCMV dr8.91, slow virus packaging plasmid pMD.2G and hNTCP
It is overexpressed plasmid pWPI-hNTCP-Puro in mass ratio 2:1:3, while be transfected into 293T cells, 48h is collected containing slow after transfection
The culture supernatant of virus, after concentration, obtains hNTCP recombinant slow virus concentrates；
Step 2：Prepare and digest complete hepatic tissue
By two step collagenase perfusion methods, digestion separation pig primary hepatocyte, obtains digesting complete hepatic tissue；
Step 3：Prepare pig primary hepatocyte
The complete hepatic tissue of digestion that step 2 is obtained, is transferred in the culture dish containing cell washing lotion, and it is thin to obtain the primary liver of pig
Born of the same parents' cell suspension；
Step 4：Prepare pig primary hepatocyte single cell suspension
The pig primary hepatocyte cell suspension that step 3 is obtained, crosses cell sieve, obtains pig primary hepatocyte single cell suspension；
Step 5：Prepare bed board cell suspension
The single cell suspension that step 4 is obtained centrifuges, and after being resuspended with cell washing lotion, centrifugation 3 times is repeated, with bed board nutrient solution weight
Outstanding cell, obtains bed board cell suspension；
Step 6：Plating cells and culture
The bed board cell suspension that step 5 is obtained is with (1.5-2.5) × 105Living cells/cm2Inoculum density, be inoculated into collagen
In coated culture plate or culture dish, bed board nutrient solution is added, is shaken up, in 37 DEG C, 5%CO2, will after cultivating 4-6h in incubator
Bed board nutrient solution changes liver cell into and maintains culture medium PMM, obtains cultured pig primary hepatocyte；
Step 7：HNTCP recombinant slow virus infection is carried out to pig primary hepatocyte
The hNTCP recombinant slow virus concentrates that step 1 is obtained, the cultured pig obtained using infection multiplicity as 1 infection step 6
Primary hepatocyte, infection are incubated 24h, change fresh liver cell into and maintain culture medium PMM, change 1 time within every 2 days, examine within the 4th day after infection
HNTCP protein expressions are surveyed, obtain the pig primary hepatocyte of hNTCP protein overexpressions；
Step 8：Establish hepatitis B virus infection cell model
In the pig primary hepatocyte of the hNTCP protein overexpressions obtained to step 7, hepatitis B virus infection blocking agent is first added, is incubated
After educating 30min, then using infection multiplicity as 1000 infection hepatitis Bs, fresh liver cell maintenance culture is changed into after infecting 16-24h
Base PMM, is replaced 1 time for every 2 days, and the 8th day detection hepatitis B virus infection index after infection, that is, obtain the utilization pig primary hepatocyte
Hepatitis B virus infection cell model is established with hNTCP recombinant slow virus.
2. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 1, the calcium phosphate transfection method is specially：Under aseptic condition, to first
450 μ L deionized waters, 50 μ L 2mol/L calcium chloride solutions, 10 μ g slow virus packaging plasmids pCMV are separately added into a centrifuge tube
Dr8.91,5 μ g slow virus packaging plasmid pMD.2G and 15 μ g hNTCP are overexpressed plasmid pWPI-hNTCP-Puro, mix and are formed
Transfection solution I；500 μ L 2 × HBS solution, side concussion are added in another centrifuge tube, side instills Transfection solution I, formed and turned
Solution II is contaminated, room temperature stands 20min；Transfection solution II is added to a diameter of 10cm, cell fusion degree 70-80%, is contained
In the 293T Tissue Culture Dish of 10mL DMEM transfection nutrient solutions, in 37 DEG C, 5%CO2After cultivating 12h in incubator, DMEM is turned
Dye nutrient solution changes fresh DMEM nutrient solutions into, i.e. transfection terminates.
3. one kind according to claim 2 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that the DMEM transfects nutrient solution, is that 10%v/v tire oxen are added in DMEM culture mediums
Serum；The fresh DMEM nutrient solutions, be added in DMEM culture mediums 100units/mL penicillin, 100mg/mL streptomysins and
10%v/v hyclones.
4. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 1, the method for the concentration is：Slow virus supernatant is first placed in 4 DEG C of water
In flat centrifuge, 3800 × g/min, centrifuges 10min, after 0.45 μm of membrane filtration, takes 15mL filtered fluids to be added to 100KD and surpass
In chimney filter, then above-mentioned 100KD super filter tubes are transferred in 4 DEG C of horizontal centrifuges again, 4000 × g/min, after centrifuging 30min,
Obtain hNTCP recombinant slow virus concentrates.
5. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 2, the two steps collagenase perfusion method, is that fresh pig liver is first
20min is irrigated by the primer solution I of 4 DEG C of precoolings, until the blood in hepatic tissue is rinsed totally, then the filling with 37 DEG C of preheatings
Solution II perfusion 30min is noted, until hepatic tissue follows the string.
6. one kind according to claim 5 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that the primer solution I is double containing 2mmol/l ethylene glycol in D-Hank ' s solution
The mixed liquor of (2- amino ethyl ethers) tetraacethyl, 7.4,0.22 μm of membrane filtration of pH value, 4 DEG C of preservations；The primer solution II is
Contain 5mmol/l calcium chloride, 0.1g/l magnesium chloride hexahydrates, 0.3g/l type Ⅳ collagenases, 1.0g/l II types in D-Hank ' s solution
Separate enzyme, the mixed liquor of 50mg/l nucleases, 7.4,0.22 μm of membrane filtration of pH value, 4 DEG C of preservations.
7. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 5, the condition of the centrifugation is 50 × g, 4 DEG C, and the time of centrifugation is
5min；The bed board nutrient solution, is ITS+premix, 2mmol/l paddy ammonia that 1%v/v is added in William ' s E culture mediums
Acid amides, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisones, 40 μ g/l dexamethasone, 100units/mL penicillin,
100mg/mL streptomysins, 5%v/v hyclones.
8. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 6, the preparation method of the coated culture plate of the collagen or culture dish is：
172 μ L glacial acetic acid are added in 100mL deionized waters, add the IV type Collagen type-I of final concentration of 50 μ g/mL, sterile bar
Under part, with 0.22 μm of membrane filtration it is degerming after, obtain collagen working solution, 4 DEG C of holdings are stand-by；By the collagen working solution with 5 μ g/
cm2Package amount be added to and need in coated culture plate or culture dish, after room temperature is incubated 1h, collagen working solution is suctioned out, from
Sealed after so drying, 4 DEG C of preservations；The liver cell maintains culture medium PMM, is to add 1%v/ in William ' s E culture mediums
ITS+premix, 2mmol/l glutamine of v, 10 μ g/l epidermal growth factor, 18mg/l hydrocortisones, fill in 40 μ g/l
Meter Song, 100units/mL penicillin, 100mg/mL streptomysins and 2%v/v DMSO.
9. one kind according to claim 1 establishes hepatitis B sense using pig primary hepatocyte and hNTCP recombinant slow virus
Contaminate the method for cell model, it is characterised in that in step 8, the concentration of the hepatitis B virus infection blocking agent is respectively
500nmol/L, 50nmol/L, 5nmol/L, 0.5nmol/L, 0.05nmol/L, 0nmol/L, the hepatitis B virus infection block
Agent is Myr-PreS12-47；The hepatitis B virus infection index is hepatitis B virus surface antigen, Hepatitis B virus e antigen and intracellular
Hepatitis B virus core antigen.
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| CN109055302A (en) * | 2018-08-28 | 2018-12-21 | 重庆医科大学附属第医院 | The method of separation, culture and the identification of HBV transgenic mice primary hepatocyte |
| CN109706181A (en) * | 2019-01-29 | 2019-05-03 | 中国农业科学院北京畜牧兽医研究所 | A kind of method, immortalization pig liver sternzellen system and application constructing immortalization pig liver sternzellen system |
| CN113684186A (en) * | 2021-09-10 | 2021-11-23 | 复旦大学 | Cell model capable of supporting hepatitis B virus replication and infection and establishing method |
| CN113755446A (en) * | 2021-07-27 | 2021-12-07 | 昆明市第一人民医院 | Method for cell model with NTCP overexpression |
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 (5)
| 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》 * |
| MING ZHOU等: "hNTCP‑expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs", 《MOL MED REP》 * |
| 勾英等: "慢病毒介导过表达NTCP的NTCP-SK-Hep1细胞系构建", 《遵义医学院学报》 * |
| 巫协宁主编: "《临床肝胆系病学》", 30 September 2002 * |
| 王文等: "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", 《病毒学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055302A (en) * | 2018-08-28 | 2018-12-21 | 重庆医科大学附属第医院 | The method of separation, culture and the identification of HBV transgenic mice primary hepatocyte |
| CN109706181A (en) * | 2019-01-29 | 2019-05-03 | 中国农业科学院北京畜牧兽医研究所 | A kind of method, immortalization pig liver sternzellen system and application constructing immortalization pig liver sternzellen system |
| CN113755446A (en) * | 2021-07-27 | 2021-12-07 | 昆明市第一人民医院 | Method for cell model with NTCP overexpression |
| CN113684186A (en) * | 2021-09-10 | 2021-11-23 | 复旦大学 | Cell model capable of supporting hepatitis B virus replication and infection and establishing method |
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