CN106676072A - Construction method of interferon-resistant HBV (Hepatitis B Virus) cell line model and application of interferon-resistant HBV cell line model in preparing anti-HBV drug - Google Patents
Construction method of interferon-resistant HBV (Hepatitis B Virus) cell line model and application of interferon-resistant HBV cell line model in preparing anti-HBV drug Download PDFInfo
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Abstract
The invention discloses a construction method of an interferon-resistant HBV (Hepatitis B Virus) cell line model and application of the interferon-resistant HBV cell line model in preparing an anti-HBV drug. The construction method comprises the following steps of cultivating an HBV in-vitro transfection cell strain HepG2.2.15; adding 10 to 70 IU.mL<-1> of low-concentration interferon IFN alpha-2b into the cell strain HepG2.2.15, and adding geneticin G418 for carrying out long-term induction for 12 to 48 weeks at the same time, thus obtaining an HepG2.2.15/IFN alpha-2b cell line model. According to the construction method of the interferon-resistant HBV cell line model, disclosed by the invention, the process route is simple, the interferon-resistant HBV cell line model is constructed, an experimental foundation is provided for a mechanism for further discovering drug resistance of HBV on interferon, and an ideal is provided for screening and developing novel anti-HBV drugs.
Description
Technical field
The present invention relates to a kind of cell induction method of hepatitis B virus, more particularly to a kind of HBV of resistance to interferon thin
The method for building up of born of the same parents system model and its application in Anti-HBV drugs are prepared.
Background technology
Interferon IFN α has antiviral and immunoregulatory dual function, is currently used for chronic hepatitis B (CHB)
One of key agents for the treatment of.The long-acting interferon developed in recent years, such as PEG-IFN α, rHSA-IFN α, overcome common interference
The shortcoming of plain half-life short, improves the compliance of patient.But either plain interferon or long-acting interferon, prolonged application
Its anti-hepatitis virus HBV, hepatitis C virus HCV effect afterwards occurs and is decreased obviously, hepatitis viruss easily to generation drug resistance, such as
After common IFN α treatment half a year, only 26% CHB patient obtains stable curative effect, and the stable curative effect rate of PEG-IFN α is also only
Have 29%.RHSA-IFN α are also imprecise to the long-term efficacy of CHB, but have HCV that the report of drug resistance is produced to rHSA-IFN α.
HBV produces the use that drug resistance has seriously limited interferonses medicine to IFN, defeats HBV to bring new challenge to the mankind.Explore
The mechanism of the resistance to interferon of HBV, and find corresponding intervening measure to delay the generation of drug resistance, will recover interferon Anti-HBV activity
The key of effect, and the HBV cell models for setting up resistance to interferon are discovery mechanism and the key for finding intervening measure.
HBV drug resistances are to limit the principal element that IFN α -2b is used, and it is research resistance mechanism to set up external mdr cell model
One of important means.The method for setting up medicine-resistant cell line at present mainly has external drug-induced method and drug resistant gene infection protocol,
Wherein drug-induced method includes that low concentration maintains method and Intermittent High-Dose impact to be stepped up dosimetry for a long time.The former is most common
Method, this method is incremental, and screening is successfully held larger, and the persister drug resistance of foundation is more stable, needs the time short.Mesh
The report of the front HBV cell models for being also not set up resistance to IFN both at home and abroad.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of HBV cell line models of resistance to interferon
Method for building up and its application in Anti-HBV drugs are prepared, set up HBV medicine-resistant cell line models.
The present invention is achieved by the following technical solutions, and the present invention is comprised the following steps：
(1) HBV in-vitro transfection Cell Line HepG2 .2.15 are cultivated；
(2) by 10~70IUmL of low concentration-1Interferon IFN α -2b be added in Cell Line HepG2 .2.15, while
Geneticin G418 is added to carry out inducing 12~48wk to obtain HepG2.2.15/IFN α -2b cell line models for a long time.
In the step (1), 80mgL is added in culture medium DMEM-1G418,10% hyclone, 2mmol/L paddy ammonia
Amide, 100IU/mL penicillin and streptomycins；PH7.2~7.4, condition of culture used are 37 DEG C, 5%CO2, after cell grows up to monolayer,
0.25% trypsinization, passes on per 3~4d once, obtains HepG2.2.15.
In the step (2), be grouped after HepG2.2.15 cell countings, be separately added into final concentration of 0 in culture fluid, 10,
30、50、70IU·mL-1IFN α -2b, while plus 380mgL-1G418 is induced, and cultivates 3~4d, is digested after cell is covered with
Counting is further cultured for, and 12~48wk of successive induction culture obtains HepG2.2.15/IFN α -2b cell line models.
Present invention additionally comprises following steps：Optimum medicine efficacy concentration is added in HepG2.2.15/IFN α -2b cell line models
Interferon IFN α -2b, determine optimal stimulus concentration and stimulation time.
The determination method of the optimum medicine efficacy concentration is as follows：
Count after taking HepG2.2.15 cell dissociations, be configured to 3 × 104Individual mL-196 well culture plates are inoculated in, after 2h, treat thin
Born of the same parents are completely adherent, give the IFN α -2b of variable concentrations respectively, and negative control group is not administered.Continue culture 96h, collect on cell
Clear liquid, the suppressed degree highest concentration of HepG2.2.15 cells secretion mark HBsAg, HBeAg, HBV DNA is optimal
Drug effect concentration.
The determination method of the optimal stimulus concentration is as follows：
Taking stimulates each group cell of 12wk, and adjustment cell concentration is 3 × 104Individual mL-1, 96 well culture plates are inoculated in, per hole
100 μ L, treat after 2h that cell is completely adherent, remove culture fluid, add the IFN α -2b culture fluid containing optimum medicine efficacy concentration, after 96h
Cell supernatant is collected, compares the change of HBsAg, HBeAg, HBV DNA in each group cell supernatant of different stimulated concentration, from
And determine optimal stimulus concentration.
The determination method of the optimal stimulus time is as follows：
Choosing above-mentioned optimal stimulus concentration stimulates the cell of different stimulated time respectively, and adjustment cell concentration is 3 × 104It is individual
mL-1, 96 well culture plates being inoculated in, per 100 μ L of hole, after 2h, treats that cell is completely adherent, remove culture fluid, addition contains optimum medicine efficacy
IFN α -2b the culture fluid of concentration, collects cell supernatant after 96h, compare in each group cell supernatant of different stimulated time
The change of HBsAg, HBeAg, HBV DNA, so that it is determined that the optimal stimulus time.
A kind of application of method for building up of the HBV cell line models of the resistance to interferon in Anti-HBV drugs are prepared.
The present invention has advantages below compared to existing technology：Present invention process route is simple, and the HBV for setting up resistance to interferon is thin
Born of the same parents' model, provides experiment basis to the mechanism that interferon produces drug resistance further to explore HBV, is the sieve of new Anti-HBV drugs
Choosing and exploitation provide thinking.
Description of the drawings
Fig. 1 is the impact that IFN α -2b secretes mark to HepG2.2.15 cells；
Fig. 2 is the impact that the IFN α -2b of optimum medicine efficacy concentration secretes mark to the cell of different stimulated concentration；
Fig. 3 is the impact that the IFN α -2b of optimum medicine efficacy concentration secretes mark to the cell of different stimulated time.
Specific embodiment
Below embodiments of the invention are elaborated, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following enforcements
Example.
The present invention medicine and reagent be：
Interferon IFN α -2b is that (Anda is fragrant, 6,000,000 IUmL for An Ke biological engineering limited company product-1), DMEM
High glucose medium, hyclone are U.S.'s Gibicol Products, and trypsin is U.S.'s Hyclone Products, hereditary mould
Plain G418 is U.S.'s Amresco Products, and ELISA enzyme linked immunological kits, HBV nucleic acid quantitative determination reagent kits are Shanghai
Ke Hua Bioisystech Co., Ltd product, remaining reagent are domestic pure analysis pure.
The detecting instrument that the present invention is used is：
Real-time quantitative PCR (qRT-PCR) instrument (Applied Biosystems companies of the U.S.), Emax Plus microplate reader,
IX51 type inverted microscopes (Japanese OLYMPUS companies), 3110 type water-jacket typ CO2Incubator (U.S. Thermo) company).
Embodiment 1
Cell culture：
HBV in-vitro transfection Cell Line HepG2 .2.15 cell lines are purchased from Shanghai Bo Dong bio tech ltd.This enforcement
Cell used by example is this cell, and culture fluid used adds 380mgL in being DMEM-1G418,10% hyclone,
2mmol/L glutamine, 100IU/mL penicillin and streptomycins, pH7.2~7.4, condition of culture used are 37 DEG C, 5%CO2, treat thin
After born of the same parents grow up to monolayer, 0.25% trypsinization is passed on once per 3~4d.
The induction of drug-resistant cell strain：
Be divided into five groups after HepG2.2.15 cell countings, be separately added in culture fluid IFN α -2b (final concentration be respectively 0,
10、30、50、70IU·mL-1), while plus G418 (380mgL-1) induction, 3~4d is cultivated, the digestion after cell is covered with is counted
It is further cultured for, according to said method successive induction culture 12 months, collects cell supernatant every month and to leave and take cultured cells frozen.
HepG2.2.15 cells be by ayw hypotypes HBV-DNA import In vitro culture human hepatoma cell line HepG2's cell and
The transfectional cell series of acquisition, can stably express whole virus markers of HBV, including DNA (deoxyribonucleic acid) HBV- of hepatitis B virus
DNA (i.e. hepatitis B virus gene), hepatitis B surface antigen HBsAg, hepatitis B virus e antigen HBeAg, hepatitis B core antigen
HBcAg etc..HepG2.2.15 cells are more ripe Anti-HBV activity research In vitro cell models, be widely used in HBV infection and medicine is anti-
The research of the aspect such as HBV effects and mechanism.
Embodiment 2
The present embodiment secretes the impact of mark for determining IFN α -2b to HepG2.2.15 cells, compares variable concentrations
IFN α -2b impacts that HBsAg, HBeAg, HBV DNA is expressed, so that it is determined that optimum medicine efficacy concentration.
Count after taking HepG2.2.15 cell dissociations, be configured to 3 × 104Individual mL-1It is inoculated in 96 well culture plates and (divides 7 groups, often
Group 6 multiple holes), treat after 2h that cell is completely adherent, give respectively variable concentrations IFN α -2b (250,500,750,1000,
1250、1500IU·mL-1), negative control group is not administered.Continue culture 96h, collect cell supernatant.
By ELISA kit description, cells and supernatant HBsAg and HBeAg detections detect that HBsAg, HBeAg exist respectively
OD values at 450nm.Standard curve is made according to standard concentration and corresponding OD values, the corresponding concentration of sample OD values is calculated,
Unit is ngmL-1.It is calculated as follows antigen suppression ratio.
Antigen suppression ratio (%)=[control wells HBsAg (or HBeAg) concentration-experimental port HBsAg (or HBeAg) concentration]/
Control wells HBsAg (or HBeAg) concentration × 100%
Cells and supernatant HBV DNA is quantitative determined：Operate according to HBV DNA fluorescence quantitative detection kits description,
Take respectively above-mentioned variable concentrations IFN α -2b (250,500,750,1000,1250,1500IUmL-1) collect after stimulation it is thin
100 μ L of born of the same parents' culture supernatant, boiling water bath 10min, 13000rpm are centrifuged 10min, take 2 μ L as pcr template, enter together with standard substance
Row real-time quantitative PCR, reaction system be 30 μ L, reaction condition：50℃2min；94℃2min；(94 DEG C of 10s, 60 DEG C of 30s) × 40
Individual circulation.Standard curve is made according to standard substance HBV DNA concentrations and corresponding Ct values, the corresponding HBV of sample Ct values is calculated
DNA concentration, unit are IUmL-1
HBV DNA suppression ratio (%)=[control wells HBV DNA concentration-experimental port HBV DNA) concentration]/control wells HBV
DNA concentration × 100%.
As shown in figure 1, HepG2.2.15 cells are Jing after the IFN α -2b of variable concentrations processes 96h, HBsAg, HBeAg and HBV
The secretion of DNA is suppressed by different degrees of, and is in dose-dependence.When the dosage of IFN α -2b is 500IUmL-1When,
Secretion to above-mentioned three marks significantly suppresses, and as dosage increases, suppresses to further enhance.When dosage is
1250IU·mL-1, HBsAg, HBeAg, HBV DNA replication dna suppression ratio be close to peak (suppression ratio be respectively 72.2%, 49.3%,
58.8%), when dosage adds to 1500IUmL-1When, HBsAg, HBeAg and HBV DNA suppression ratio increases are slower.Therefore can recognize
For 1250IUmL-1For optimum medicine efficacy concentration.
Other embodiment is identical with embodiment 1.
Embodiment 3
The present embodiment is used to determine optimal irritaiting concentration.
Choosing stimulates each group cell of 12wk, and adjustment cell concentration is 3 × 104Individual mL-1, 96 well culture plates are inoculated in, often
100 μ L of hole, treat after 2h that cell is completely adherent, remove culture fluid, the IFN containing optimum medicine efficacy concentration for adding embodiment 2 to determine
α -2b culture fluid, negative control group are not added with any medicine, and 6 multiple holes are set per group, and cell supernatant is collected after 96h, and comparison is different
The change of HBsAg, HBeAg, HBV DNA in each group cell supernatant of irritaiting concentration, so that it is determined that optimal stimulus concentration.
Using 17.0 statistical softwares of SPSS, measurement data withRepresent, multigroup is compared using single factor test variance point
Analysis, it is statistically significant as difference with P ＜ 0.05.
1st, the inhibitory action that IFN α -2b is expressed to each group cell HBsAg
As a result show, have six groups, wherein the 1st group be non-administered group, administration group secretion HBsAg be substantially less than non-administration
Group (the 1st group) (p<0.01) IFN α -2b (1250IUmL, are shown-1) expression of HBsAg can be significantly inhibited.Stimulation group (the 3rd, 4,
5th, 6 groups) HBsAg that secretes is not apparently higher than stimulating group (the 2nd group) (p<0.01), show Jing low concentration IFN αs -2b (respectively
10、30、50、70IU·mL-1) after stimulation, HBsAg is to IFN α -2b (1250IUmL-1) sensitivity reduce.Compare between each group
Compared with 30IUmL-1Group and 50IUmL-1Group suppression ratio is significantly lower than 10IUmL-1Group and 70IUmL-1Group (p<0.05), its
Middle 50IUmL-1Group suppression ratio is significantly lower than 30IUmL-1Group (P<0.01).It is shown in Table 1.
Inhibitory action that 1 IFN α -2b of table is expressed to each group cell HBsAg (N=6)
Note：Compare with the 1st group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01；Compare with 4 groups：3)3)P<0.01,3)P<
0.05；Compare with 5 groups：4)4)P<0.01
2nd, the inhibitory action that IFN α -2b is expressed to each group cell HBeAg
As a result show, the HBeAg of administration group secretion is substantially less than non-administered group (the 1st group) (p<0.01), show IFN α -2b
(1250IU·mL-1) can significantly inhibit the expression of HBeAg, the HBeAg that stimulation group (the 4th, the 5th group) is secreted is not apparently higher than stimulating
Group (the 2nd group) (p<0.05), show Jing low concentration IFN α -2b (respectively 30,50IUmL-1) after stimulation, HBeAg to IFN α-
2b(1250IU·mL-1) sensitivity reduce.Compare between each group, 30IUmL-1Group and 50IUmL-1Group suppression ratio is quite, poor
Different not statistically significant (P>0.05).It is shown in Table 2.
Inhibitory action that 2 IFN α -2b of table is expressed to each group cell HBeAg (N=6)
Note：Compare with 1 group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01,2)P<0.05；Compare with 4 groups：3)P<0.05；
Compare with 5 groups：4)4)P<0.01
3rd, the inhibitory action that IFN α -2b is expressed to each group cell HBV DNA
As a result show, the HBV DNA of administration group secretion are substantially less than non-administered group (the 1st group) (p<0.01), show IFN α-
2b(1250IU·mL-1) expression of HBV DNA can be significantly inhibited.The HBV DNA that stimulation group (the 4th, 5,6 groups) is secreted apparently higher than
Group (the 2nd group) (p is not stimulated<0.01), show Jing low concentration IFN α -2b (respectively 30,50,70IUmL-1) after stimulation, HBV
DNA is to IFN α -2b (1250IUmL-1) sensitivity reduce.Compare between each group, 50IUmL-1Group and 70IUmL-1Group suppression
Rate processed is significantly lower than 30IUmL-1Group (p<0.01), wherein 50IUmL-1Group and 70IUmL-1Group suppression ratio is suitable, difference
Not statistically significant (P>0.05).It is shown in Table 3.
Inhibitory action that 3 IFN α -2b of table is expressed to each group cell HBV DNA (N=6)
Note：Compare with 1 group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01；Compare with 4 groups：3)3)P<0.01；With 5
Group compares：4)4)P<0.01
As shown in Fig. 2 Jing 50IUmL-1After IFN α -2b stimulates, HBsAg, HBeAg, HBV DNA suppression ratio declines most bright
It is aobvious, compare with compared with control cells and reduce 25.48%, 8.40%, 15.43% respectively.Therefore it is believed that 50IUmL-1For optimal stimulus
Concentration.
Other embodiment is identical with embodiment 2.
Embodiment 4
The present embodiment is used to determine optimal stimulation time that selection embodiment 3 obtains optimal stimulus concentration to stimulate respectively
The cell of 12wk, 18wk, 24wk, 30wk, 36wk, 42wk, 48wk, by the method for above-described embodiment 3, compares the different stimulated time
Each group cell supernatant in HBsAg, HBeAg, HBV DNA change, so that it is determined that the optimal stimulus time.
1st, the inhibitory action that IFN α -2b is expressed to each group cell HBsAg
As a result show, the HBsAg of administration group secretion is substantially less than non-administered group (the 1st group) (p<0.01), show IFN α -2b
(1250IU·mL-1) expression of HBsAg can be significantly inhibited.18wk~48wk each group cell secretion HBsAg apparently higher than
12wk (the 2nd group) (p<0.05) Jing 50IUmL, are shown-1IFN α -2b stimulates 18wk~48wk, and HBsAg is to IFN α -2b
(1250IU·mL-1) sensitivity reduce.Compare between each group, Jing 50IUmL-1IFN α -2b stimulates 12wk~36wk, HBsAg
Suppression ratio declines obvious, the statistically significant (p of difference<0.05), continue to stimulate to 48wk, 6 groups are compared with 7,8 groups, difference
Not statistically significant (p>0.05) Jing 50IUmL, are shown-1IFN α -2b stimulates 36wk, and HBsAg suppression ratio is reduced to minimum.See
Table 4.
Inhibitory action that 4 IFN α -2b of table is expressed to each group cell HBsAg (N=6)
Note：Compare with 1 group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01,2) P<0.05；Compare with 5 groups：3)3)P
<0.01,3) P<0.05；Compare with 6 groups：4)4)P<0.01
2nd, the inhibitory action that IFN α -2b is expressed to each group cell HBeAg
As a result show, the HBeAg of administration group secretion is substantially less than non-administered group (the 1st group) (p<0.01), show IFN α -2b
(1250IU·mL-1) expression of HBsAg can be significantly inhibited.The HBeAg of 24wk~48wk each groups cell secretion is apparently higher than 12wk
(the 2nd group) (p<0.05) Jing 50IUmL, are shown-1IFN α -2b stimulates 24wk~48wk, and HBeAg is to IFN α -2b (1250IU
mL-1) sensitivity reduce.Compare between each group, Jing 50IUmL-1IFN α -2b stimulates 24wk~30wk, HBsAg suppression ratio to decline
Substantially, the statistically significant (p of difference<0.05), continue to stimulate to 48wk, 5 groups are compared with 6,7,8 groups, no statistical difference
Meaning (p>0.05) Jing 50IUmL, are shown-1IFN α -2b stimulate 30wk after, HBeAg suppression ratio is no longer reduced.It is shown in Table 5.
Inhibitory action that 5 IFN α -2b of table is expressed to each group cell HBeAg (N=6)
Note：Compare with 1 group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01,2) P<0.05；Compare with 5 groups：3)3)P
<0.01,3) P<0.05；Compare with 6 groups：4)4)P<0.01,4) P<0.05
3rd, the inhibitory action that IFN α -2b is expressed to each group cell HBV DNA
As a result show, the HBV DNA of administration group secretion are substantially less than non-administered group (the 1st group) (p<0.01), show IFN α-
2b(1250IU·mL-1) expression of HBV DNA can be significantly inhibited.The HBV DNA of 18wk~48wk each groups cell secretion are substantially high
In 12wk (the 2nd group) (p<0.05) Jing 50IUmL, are shown-1IFN α -2b stimulates 18wk~48wk, and HBV DNA are to IFN α -2b
(1250IU·mL-1) sensitivity reduce.Compare between each group, Jing 50IUmL-1IFN α -2b stimulates 12wk~36wk, HBsAg
Suppression ratio declines obvious, the statistically significant (p of difference<0.01), continue stimulate to 48wk, 6 groups are compared with 7 groups, difference without
Statistical significance (p>0.05), 6 groups are compared with 8 groups, and 36wk HBV DNA suppression ratio is less than 48wk, the statistically significant (p of difference
<0.05).Show Jing 50IUmL-1IFN α -2b stimulate 36wk after, HBV DNA suppression ratio reaches minimum.It is shown in Table 6.
Inhibitory action that 6 IFN α -2b of table is expressed to each group cell HBV DNA (N=6)
Note：Compare with 1 group：1)1)P<0.01；Compare with 2 groups：2)2)P<0.01；Compare with 5 groups：3)3)P<0.01；With 6
Group compares：4)4)P<0.01
As shown in figure 3, Jing 50IUmL-1IFN α -2b stimulate 36wk after, under HBsAg, HBeAg, HBV DNA suppression ratio
Drop most substantially, is compared with compared with control cells, and suppression ratio reduces 38.64%, 15.71%, 30.17% respectively, therefore is believed that 36wk
For the optimal stimulus time.
Other embodiment is identical with embodiment 3.
The present invention is by HepG2.2.15 cell lines low concentration IFN α -2b (10~70IUmL-1) continued stimuluses, while plus
Enter G418 (380mgL-1) induction, as a result show, after stimulating 12 weeks, 50IUmL-1Group cell is to optimum medicine efficacy concentration
The sensitivity of IFN α -2b is significantly reduced, and declining in various degree occurs in HBsAg, HBeAg, HBV DNA suppression ratio, has generation drug resistance
The trend of property；Continue to stimulate near minimum to suppression ratio after 36 weeks, show the cells resistance degree highest under this state.
The present invention it is variant with the method for building up of HCV mdr cell models, the time of induction is longer, this may with not
It is relevant with cell strain and viral this body structure.HBV belongs to Hepadnaviridae, is the single-stranded circular double stranded DNA in part, and third
Hepatitis virus are single strand plus RNA virus, are made up of the noncoding region of an open reading frame and both sides.
RNA viruses are more easy to mutation occur compared with DNA, therefore speculate, HBV is more conservative compared with HCV, and mutation occurs in gene order
Probability it is more lower.
New Anti-HBV drugs can be prepared on the basis of the HepG2.2.15/IFN α -2b mdr cell models of the present invention.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (8)
1. a kind of method for building up of the HBV cell line models of resistance to interferon, it is characterised in that comprise the following steps：
(1) HBV in-vitro transfection Cell Line HepG2 .2.15 are cultivated；
(2) by 10~70IUmL of low concentration-1Interferon IFN α -2b be added in Cell Line HepG2 .2.15, while add
Geneticin G418 carries out inducing 12~48wk to obtain HepG2.2.15/IFN α -2b cell line models for a long time.
2. a kind of method for building up of the HBV cell line models of resistance to interferon according to claim 1, it is characterised in that institute
State in step (1), 80mgL is added in culture medium DMEM-1G418,10% hyclone, 2mmol/L glutamine,
100IU/mL penicillin and streptomycins；PH7.2~7.4, condition of culture used are 37 DEG C, 5%CO2, after cell grows up to monolayer,
0.25% trypsinization, passes on per 3~4d once, obtains HepG2.2.15.
3. a kind of method for building up of the HBV cell line models of resistance to interferon according to claim 1, it is characterised in that institute
State in step (2), be grouped after HepG2.2.15 cell countings, be separately added into final concentration of 0 in culture fluid, 10,30,50,
70IU·mL-1IFN α -2b, while plus 380mgL-1G418 is induced, and cultivates 3~4d, and the digestion after cell is covered with is counted again
Culture, 12~48wk of successive induction culture obtain HepG2.2.15/IFN α -2b cell line models.
4. the method for building up of the HBV cell line models of a kind of resistance to interferon according to claim 1, it is characterised in that also
Comprise the following steps：Interferon IFN α-the 2b of optimum medicine efficacy concentration is added in HepG2.2.15/IFN α -2b cell line models,
Determine optimal stimulus concentration and stimulation time.
5. a kind of method for building up of the HBV cell line models of resistance to interferon according to claim 4, it is characterised in that institute
The determination method for stating optimum medicine efficacy concentration is as follows：
Count after taking HepG2.2.15 cell dissociations, be configured to 3 × 104Individual mL-196 well culture plates are inoculated in, after 2h, treat that cell is complete
It is complete adherent, the IFN α -2b of variable concentrations is given respectively, negative control group is not administered.Continue culture 96h, collect cell supernatant,
The suppressed degree highest concentration of HepG2.2.15 cells secretion mark HBsAg, HBeAg, HBV DNA is that optimum medicine efficacy is dense
Degree.
6. a kind of method for building up of the HBV cell line models of resistance to interferon according to claim 5, it is characterised in that institute
The determination method for stating optimal stimulus concentration is as follows：
Taking stimulates each group cell of 12wk, and adjustment cell concentration is 3 × 104Individual mL-1, 96 well culture plates are inoculated in, per 100 μ of hole
Treat after L, 2h that cell is completely adherent, remove culture fluid, add the IFN α -2b culture fluid containing optimum medicine efficacy concentration, collect after 96h
Cell supernatant, compares the change of HBsAg, HBeAg, HBV DNA in each group cell supernatant of different stimulated concentration, so as to true
Determine optimal stimulus concentration.
7. a kind of method for building up of the HBV cell line models of resistance to interferon according to claim 6, it is characterised in that institute
The determination method for stating the optimal stimulus time is as follows：
Choosing above-mentioned optimal stimulus concentration stimulates the cell of different stimulated time respectively, and adjustment cell concentration is 3 × 104Individual mL-1,
96 well culture plates are inoculated in, per 100 μ L of hole, after 2h, treat that cell is completely adherent, remove culture fluid, added containing optimum medicine efficacy concentration
IFN α -2b culture fluid, cell supernatant is collected after 96h, compare HBsAg in each group cell supernatant of different stimulated time,
The change of HBeAg, HBV DNA, so that it is determined that the optimal stimulus time.
8. a kind of method for building up of the HBV cell line models of resistance to interferon as claimed in claim 1 is in Anti-HBV drugs are prepared
Application.
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Non-Patent Citations (4)
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| NAMBA K等: "Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype", 《BIOCHEMICAL & BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 宁琴等: "《乙型肝炎重症化基础与临床》", 31 January 2014, 华中科技大学出版社 * |
| 徐丙发等: "干扰素-α及长效干扰素抗肝炎病毒作用机制的研究进展", 《中国药理学通报》 * |
| 谢霞等: "HepG2.2.15细胞系中HBV复制相关抑制因素的变化", 《实用医学杂志》 * |
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