The Establishment Of Screening-evaluation System For Anti-hepatitis B Virus Drugs And The Empirical Study Of Gankang Suppository On Hepatitis B Virus

【Objective】To establish a real-time fluorescent quantitation PCR system using Taqman probe fordetecting hepatitis B virus gene.The HepG2.2.15 cell strain and duck hepatitis B animalmodel were used to study hepatitis B virus respectively in vitro or in vitro.Accordingly,thescreening-evaluation system for anti-hepatitis B virus drugs was established.The effect ofGankang Suppository on hepatitis B virus in vitro and in vivo was studied utilizing thissystem.【Methods】1.According to the characters of viral genome and nucleotide sequence,3 special primerpairs and Taqman probe were designed.The target gene fragments of HBV DNA,DHBVDNA and DHBV cccDNA were amplified and were cloned in pUCm-T vector after PCRproducts purification and gel extraction,and the plasmids were transformed into DH5αstrains.The recombinant plasmids evaluated by PCR and sequencing were prepared for standardsubstance and standard curve of real-time quantitation PCR.Optimizing the reaction systemand parameter,the real-time fluorescent quantitation PCR system using Taqman probe fordetecting hepatitis B virus gene was established.The sensibility,specificity and repeatabilityof this system were estimated.2.The growth status and HBsAg,HBeAg secretion of HepG2.2.15 were observed afterculturing 1,2,4,6,8,10,12,14d.The cytotoxic effect of Gankang Suppository and lamivudine were detected by MTT assay,and the 50% Toxic Concentrations wereaccounted.The HBsAg,HBeAg,HBV DNA in supernate were detected by ELISA and PCRon 4^th,7^th,10^thday after adding Gankang Suppository (12mg/ml,10mg/ml,6mg/ml and5mg/ml)and lamivudine (0.2μg/ml,1μg/ml,5μg/ml and 25μg/ml).The 50% EffectiveConcentration and Selection Index of Gankang Suppository and lamivudine to HBsAg,HBeAg,HBV DNA in supernate on 10^thday were accounted.3.The DHBV DNA extracted from 244 serum of adult Yingtao Gu duck in Wuhan wasdetected by PCR and agarose gel electrophoresis and the DHBV DNA positive rate wasestimated.The congenital DHBV DNA(-)ducklings picked from 96 Yingtao Gu ducklingsby PCR were used to prepared animal model and the DHBV natural infection rate of YingtaoGu ducklings was also estimated.The method of DHBV DNA positive serum injection(0.2ml)via vena cruralis was used to prepare postnatal infection duck hepatitis B virus animal model,and the DHBV infection rate was accouted.4.The normal Yingtao Gu ducklings were used to study short term toxicity effect ofGankang Suppository.The ducklings of postnatal infectious duck hepatitis B virus weredivided randomly to:control group,ACV control group,Gankang Suppository large-dosegroup,medium-dose group,small-dose group,(12 ducklings in every group),and 12 twoweeks old normal ducklings were selected for normal control group.The DHBV DNA,ALTand AST were respectively detected before treatment (T₀),14 days (T₁₄)and 28 days (T₂₈)after treatment,and 7 days after withdrawal (P₇).The histopathological change,DHBV DNA,and DHBV cccDNA of liver tissue was observed 7 days after withdrawal.【Results】1.The three standard substances of pUCm-HBV DNA,pUCm-DHBV DNA andpUCm-DHBV cccDNA were successfully constructed.In the range of (1×10²ï½ž1×10⁹)copies/ml,the coefficient correlation (r)of these standard substances content to Ct were:-1.00,-0.999 and-0.997 respective.In the range of (5×10³ï½ž5×10⁸)copies/ml,the Ct coefficient of variability of these standard substances were all less than 5%,which weredetected repetitively for 4 times in the same instrument for real-time PCR.The sensibilities ofdetecting HBV DNA,DHBV DNA and DHBV cccDNA used this real-time fluorescentquantitation PCR system were 5×10¹copies/ml,5×10¹copies/ml and 1×10²copies/ml.Thepositive amplification was detected in HBV DNA positive supernate,DHBV DNA positiveduck serum and DHBV cccDNA positive hepatic tissue,conversely in blank culture media,normal duck serum and hepatic tissue.2.â‘ HepG2.2.15 cell growth best on 8^th-12^thday after microbiomation.The HBsAg andHBeAg in supernate were positive on 1st day,and the secretory volume of HBsAg andHBeAg increased gradually along with culture time lapsing,and achieved peak on 10^thday.Even though the increasing velocity of HBsAg secretory volume bigger than HBeAg in 1^st-6^th,the secretory volume of HBeAg always more than HBsAg in the same period duringwhole culture.â‘¡In the range of (7.5～60)mg/ml Gankang Suppository,the inhibitory effect toHepG2.2.15 cell multiplication enhanced gradually along with drug concentration increasing.And when the concentration of Gankang Suppository was small than 15mg/ml,the inhibitoryrate of HepG2.2.15 cell multiplication was small than 20%.â‘¢The inhibitory rate of HBsAg and HBeAg on 10th day after adding 12mg/mlGankang Suppository were the greatest:68.2% and 62.7%.It showed apparent time and dosedependent.And the inhibitory rate of HBsAg and HBeAg on 7^thday after adding 25μg/ml3TC were the greatest:50.1% and 47.3%,and the inhibitory rate of HBsAg and HBeAg on10^thday was lower than that on 7^thday,and did not show time and dose dependent.â‘£The inhibitory rate of HBV DNA of supernate was greatest (78.8%)on 10^thday afteradding 12mg/ml Gankang Suppository.However,The inhibitory rate of HBV DNA ofsupernate was greatest (97.9%)on 10^thday after adding 25μg/ml 3TC.⑤The selection index of Gankang Suppository and 3TC were 6.14 and 31450.5.3.There were 52 DHBV DNA positive sera in 244 sera of adult Yingtao Gu duck collected in Wuhan,and the DHBV DNA positive rate in adult Yingtao Gu duck was[16.64%～26.87%] (95% confidence intervals).There were only 11 DHBV DNA positiveducklings in 96 one day old Yingtao Gu ducklings,detected before injecting DHBV DNAserum.The DHBV DNA natural infection rate of Yingtao Gu ducklings was [6.52%～19.36%] (95% confidence intervals).In 80 one day old Yingtao Gu ducklings randomselected to prepare model,69 ducklings were DHBV DNA positive after 1 week of injectingDHBV DNA positive serum.The DHBV infectious rate was 86.25%.4.â‘ Before treatment,7 days,14 days and 28 days after treatment,no significantdifferences were found among the Gankang Suppository large-dose group,medium-dosegroup,small-dose group and control group,in terms of feather color/luster,gait,food-taking,mental status and response to stimuli,and no significant differences were found among everygroups in every experimental stages,in terms of body weight and weight gain.(P＞0.05)â‘¡The DHBV DNA level in Gankang Suppository large-dose group was always lowerthan other groups in the same stage,and it descended to the minimum on P₇ (P＜0.01).TheDHBV DNA level in Gankang Suppository large-dose group and medium-dose group had notrebounded on P₇,however,the DHBV DNA level in Gankang Suppository small-dose groupdid not rebound had rebounded.â‘¢The DHBV DNA and DHBV cccDNA of hepatic tissue in the Gankang Suppositorylarge-dose group had significantly decreased on P₇(P＜0.05).â‘£Compared with model control group,the ALT values of Gankang Suppositorylarge-dose group,medium-dose group were significantly lower on T₂₈and P₇ (P＜0.01),andthe AST value in Gankang Suppository large-dose group was also significantly lower on T₁₄(P＜0.05).Compared with ACV group,the ALT level of Gankang Suppository large-dosegroup had significantly decreased at T₂₈(P＜0.05),and the ALT level of Gankang Suppositorylarge-dose group,medium-dose group had significantly decreased at P₇(P＜0.05).However,the ALT and AST level of ACV control group had not significantly decreased during thewhole treatment. ⑤Histopathological examination of the liver showed:Compared with DHBV modelcontrol group,the inflammatory cell infiltrate and necrosis in Gankang Suppositorylarge-dose group,medium-dose group and small-dose group were all alleviated,especially inlarge-dose group and medium-dose group.【Conclusion】1.In the study,a real-time fluorescent quantitation PCR system using Taqman probe fordetecting hepatitis B virus gene was successfully established,and sensibility,specificity andrepeatability of this system were very well estimated by methodology.A screening-evaluationsystem for anti-hepatitis B virus drugs was established in principle.2.The drug study was condign to carry out on 8^th-10^thof HepG2.2.15 cell culture,inwhich phase the cell was in summit of growth.The cytotoxicity of Gankang Suppository wasslight in vitro,and it can inhibit the secretion of HBsAg,HBeAg and HBV DNA certainly.3.The Yingtao Gu duckling was applied to prepare duck hepatitis B animal model byDHBV injection,because the DHBV infection rate in Yingtao Gu duck in Wuhan and theDHBV natural infection rate in the duckling were both low.The Yingtao Gu ducklinghepatitis B model infected postnatal DHBV was successfully prepared.4.Gankang Suppository had no obvious short-term toxicity to ducklings,it prompted thatGankang Suppository is safe for treating hepatitis B.It was dose and time dependent thatGankang Suppository inhibited the DHBV DNA level in model ducklings.GankangSuppository could effectively suppress the DHBV DNA and DHBV cccDNA in hepatic tissue.Gankang Suppository could also improve the serum biochemistry and liver histopathology.Itwas considered that the efficacy of Gankang Suppository may does not only result from itspotent suppression of DHBV replication,but be relation to its other active component(glycyrrchizin,lentinan).All of these above,it is indicated sufficiently that the compoundpreparation of Gankang Suppository had some advantage in treatment of hepatitis B.