【作者】 李晖；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 【目的】建立一套检测乙型肝炎病毒基因的Taqman探针实时荧光定量PCR系统、分别利用HepG2．2．15细胞模型和鸭乙型肝炎模型于体外、体内研究乙型肝炎病毒，从而建立并完善抗乙型肝炎病毒药物筛选、评价体系。并利用该体系评价肝康栓体内、外抗乙型肝炎病毒的作用。【方法】1．根据病毒基因组和核苷酸序列特点，分别设计3对特异性引物及Taqman探针。利用这3对引物分别扩增HBV DNA、DHBV DNA及DHBV cccDNA目的基因片段。PCR产物纯化、回收后，克隆入pUCm-T载体，转化DH5α菌株，筛选阳性菌落，提取重组质粒。重组质粒经PCR及测序鉴定后，用于制备实时荧光定量PCR的标准品和绘制标准曲线。通过优化反应体系和循环参数，建立乙型肝炎病毒基因Taqman探针实时荧光定量PCR检测系统。并进一步评价该系统的灵敏度、特异度及可重复性。2．体外培养HepG2．2．15细胞，观察该细胞培养1、2、4、6、8、10、12、14天时的生长情况和HBsAg、HBeAg的分泌情况。用MTT法检测肝康栓和拉米夫定对HepG2．2．1 5细胞的细胞毒性作用，计算肝康栓和拉米夫定的半数中毒浓度。并分别用ELISA法或PCR法检测肝康栓（12mg／ml、10mg／ml、6mg／ml和5mg／ml的终浓度）与拉米夫定（0．2μg／ml、1μg／ml、5μg／ml和25μg／ml的终浓度）作用4、7、10天时，HepG2．2．15细胞培养上清液中HBsAg、HBeAg、HBV DNA的抑制作用。计算培养第10天时肝康栓和拉米夫定抑制HepG2．2．15细胞上清液中HBsAg、HBeAg分泌及HBV DNA复制的半数有效浓度。最后评价肝康栓的选择指数。3．分别提取244份武汉地区成年樱桃谷鸭血清中的DHBV DNA，并进行PCR扩增和琼脂糖凝胶电泳。估计武汉地区成年樱桃谷鸭DHBV携带率。取1日龄樱桃谷雏鸭96只，利用PCR法筛选先天DHBV DNA（-）雏鸭用于制备动物模型，并估计樱桃谷雏鸭DHBV自然感染率。通过胫静脉注射DHBVDNA强阳性血清（0．2ml／只），利用先天DHBV DNA（-）雏鸭制备后天感染鸭乙型肝炎病毒动物模型，并计算DHBV造模感染率。4．采用正常樱桃谷雏鸭研究肝康栓短期毒性反应。将后天感染鸭乙型肝炎病毒动物模型雏鸭随机分为：模型对照组，ACV对照组，大、中、小剂量肝康栓治疗组，每组12只；再取12只2周龄的正常雏鸭作为正常对照组。分别于第一次给药前（T₀）、给药14d(T₁₄)、给药28d(T₂₈)和停药7d（P₇）时，胫静脉取血（0．5ml／只），分离血清，检测肝康栓对雏鸭血清中DHBVDNA的抑制情况和血清ALT、AST的影响。并于停药7d时将各组鸭处死，统一取肝右叶2块组织，分别用于检测肝脏DHBV DNA、DHBVcccDNA和组织病理学。【结果】1．本实验成功构建了pUCm-HBV DNA、pUCm-DHBV DNA和pUCm-DHBVcccDNA三种质粒标准品。在（1×10²～1×10⁹）copies／ml浓度范围内，三种标准品的含量与Ct值的相关系数r分别为-1．00、-0．999和-0．997。（5×10³～5×10⁸）copies／ml 6个浓度的三种质粒标准品，在同一实时荧光定量PCR仪上重复进行4次扩增，Ct值的变异系数均小于5％。利用本实验所建立的Taqman探针实时荧光定量PCR系统检测HBV DNA、DHBV DNA和DHBV cccDNA的灵敏度分别为：5×10¹ copies／ml、5×10¹ copies／ml和1×10²copies／ml；并且HBV DNA阳性上清液、DHBV DNA阳性鸭血清和DHBV cccDNA阳性鸭肝组织用本法检测均出现阳性扩增，而空白的培养基、正常鸭血清及正常鸭肝组织均未出现阳性扩增。2．①HepG2．2．15细胞按1×10⁶／L浓度接种、培养后，第8～12天细胞生长状态最佳；并且用ELISA法检测培养上清发现培养1天后，检测细胞上清液中的HBsAg和HBeAg均为阳性，其后培养上清液中HBsAg及HBeAg的分泌量随着培养时间的增长而逐渐增加，在培养第10天达到高峰。培养第1-6天时HBsAg分泌增长速度较HBeAg快；但是整个培养过程中，HBeAg分泌量均高于同时期HBsAg的分泌量。②肝康栓在（7．5～60）mg／ml浓度范围内，对HepG2．2．15细胞增殖的抑制作用随肝康栓的浓度增加而增强，当肝康栓浓度小于15mg／ml时，对细胞增殖的抑制率＜20%。③12mg／ml的肝康栓作用10天后，HBsAg、HBeAg的抑制率达最大，分别为68．2％和62．7％。呈明显的时间和剂量依赖性；而3TC在25μg／ml浓度下作用7天，HBsAg、HBeAg的抑制率最大，分别为50．1％和47．3％。且3TC培养10天时，HBsAg、HBeAg的抑制率均较培养7天时减弱。无明显的时间依赖性。④肝康栓在12mg/ml浓度、作用10天时，对HepG2．2．15细胞上清中HBV DNA的抑制率较强，为78．8％；而3TC在25μg／ml浓度、作用10天时，对HepG2．2．15细胞上清中HBV DNA的抑制率最强，为97．9%。⑤肝康栓和3TC抑制HBV DNA的选择指数分别为6．14和31450．5。3．在244份武汉地区采集的成年樱桃谷鸭血清标本中，52份为DHBV DNA阳性血清，估计武汉地区成年樱桃谷鸭DHBV携带率为：[16．64％～26．87%]（95％置信区间）。而96只1日龄樱桃谷雏鸭中，在接种前检测DHBV DNA，只有11只为DHBV DNA阳性，估计樱桃谷雏鸭DHBV自然感染率为[6．52％～19．36%]（95％置信区间）。随机选取1日龄DHBV DNA（-）的樱桃谷雏鸭80只，接种DHBV DNA强阳性血清1周后，69只为DHBV DNA阳性，8只为阴性，3只死亡，DHBV造模感染率为86．25%。4．①肝康栓大、中、小剂量组雏鸭给药前、给药7天、给药14天、给药28天时，与对照组雏鸭相比，在羽毛色泽，步态，进食状况，精神状态，对刺激的反应等方面也均无明显差异；各组雏鸭在试验各阶段，体重及体重增重值均无统计学差异（p＞0．05）。②肝康栓大剂量组雏鸭血清DHBV DNA始终低于同期其他组，且在停药7天时下降至最低（p＜0．01）；肝康栓大、中剂量组在停药7天后DHBV DBA均未出现反弹，而肝康栓小剂量组及ACV对照组均有不同程度的反弹。③停药7天时，肝康栓大剂量组雏鸭肝组织DHBV DNA和DHBV cccDNA均受明显抑制，结果有统计学意义（p＜0．05）。④与同期模型组比较，肝康栓中、大剂量组雏鸭在治疗28天和停药7天时血清ALT明显降低，有显著差异（p＜0．01）。肝康栓大剂量组雏鸭在治疗14天时血清AST也明显降低，有显著差异（p＜0．05）；与同期ACV对照组比较，肝康栓大剂量组在给药28天时，血清ALT明显降低，有显著差异（p＜0．05）。停药7天时，肝康栓中、大剂量组ALT也明显降低，有显著差异（p＜0．05）。而ACV对照组在治疗过程中ALT、AST均未见显著下降。⑤肝组织病理检查结果显示：肝康栓小、中、大剂量组与模型组相比，汇管区炎性细胞浸润和肝细胞点、灶状坏死均有不同程度的改善。其中肝康栓大、中剂量组改善较为明显。【结论】1．本实验成功构建了一套检测乙型肝炎病毒基因的Taqman探针实时荧光定量PCR系统，并且通过方法学评价该系统具有很好的灵敏度、特异度和可重复性。基本建立了一套较完善的抗乙型肝炎病毒药物筛选、评价体系。2．培养第8～12天的HepG2．2．15细胞处于生长高峰期，在此期进行药物实验较适宜。肝康栓体外实验的细胞毒性较小，并且对HepG2．2．15细胞培养上清液中HBsAg、HBeAg及HBV DNA有一定的抑制作用。3．武汉地区成年樱桃谷鸭的DHBV携带率不高，其雏鸭DHBV自然感染率较低，适用于制备感染DHBV的鸭乙型肝炎动物模型。并且在本研究中成功制备了后天感染DHBV的樱桃谷雏鸭模型。4．肝康栓对雏鸭无明显短期毒性作用，初步提示肝康栓作为治疗慢性乙型肝炎的药物是安全的。肝康栓能抑制模型雏鸭血清中的DHBV DNA，且存在一定的剂量和时间依赖性。肝康栓能有效抑制模型雏鸭肝组织中的DHBV DNA和DHBV cccDNA。肝康栓还能改善雏鸭的血清生化学和肝组织病理学。我们认为这些作用除了与肝康栓能有效抑制DHBV DNA的复制外，还与其组方中的有效成分（甘草甜素、香菇菌多糖）有关。这也充分显示了肝康栓作为复方制剂治疗慢性乙型肝炎的优势。更多还原

【Abstract】 【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 （1×10²～1×10⁹）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 （5×10³～5×10⁸）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.2.①HepG2.2.15 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.②In 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%.③The 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.④The 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%.4.①Before 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）②The 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.③The DHBV DNA and DHBV cccDNA of hepatic tissue in the Gankang Suppositorylarge-dose group had significantly decreased on P₇（P＜0.05）.④Compared 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.更多还原