CAJ | 학술논문

目的通过体内转导乙型肝炎病毒(HBV) cccDNA,建立持续表达HBV抗原的小鼠乙型肝炎模型. 方法取10只裸小鼠,将HBV cccDNA以3μg/只、2 μg/只、1μg/只分别注射实验组,同时以2ml等渗盐水注射空白组,分别于第1、3天,第1、2、3、4、5、6、8、10周进行尾静脉采血.用放射免疫法检测血清中乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)浓度的变化;荧光定量PCR检测血清和肝组织中HBV DNA拷贝数;免疫组织化学法检测肝组织中HBV抗原特异性;苏木素-伊红(HE)染色观察肝组织病理变化.使用SPSS 17.0对数据间相关性进行线性相关分析. 结果小鼠在注射cccDNA后,从第1天开始于血液中检测到HBsAg和HBeAg的表达,两者均于第1周内迅速升高达到最高值,并持续至第10周;其中HBsAg的表达水平经历了两个先上升再下降的过程(第4周为较低水平,第8周为较高水平),而HBeAg的表达水平经历了三个先上升再下降的过程(第2周、第4周为较低水平,第3周、第6周为较高水平);荧光定量PCR结果表明,10周后肝组织中HBV DNA的拷贝数随着高、中、低剂量组而递减,每克肝组织中HBV DNA拷贝数[(1.14E+07)±(6.51E+06)、(9.81E+06)±(9.32E+06)、(3.72E+06)±(2.35E+ 06)]与注射的cccDNA浓度(1.5、1.0、0.5 μg/ml)呈正相关(皮尔森相关系数r=0.979).10周后血清中HBV DNA的拷贝数为中剂量组最高,其次为高剂量组,同时,各组血清中的HBV DNA拷贝数较肝组织中的低;免疫组织化学结果显示,实验组裸小鼠肝组织中存在HBsAg和HBcAg的表达,HE染色结果显示肝组织脂肪样或空泡样变性严重,肝小叶组织结构不明显. 结论本研究利用HBVcccDNA质粒体内转导裸小鼠,成功建立了HBV在肝组织稳定复制并持续表达HBV抗原的裸小鼠模型,为进一步研究HBVcccDNA慢性持续感染的机制和抗病毒治疗打下基础.
목적 통과체내전도을형간염병독(HBV) cccDNA,건립지속표체HBV항원적소서을형간염모형. 방법 취10지라소서,장HBV cccDNA이3μg/지、2 μg/지、1μg/지분별주사실험조,동시이2ml등삼염수주사공백조,분별우제1、3천,제1、2、3、4、5、6、8、10주진행미정맥채혈.용방사면역법검측혈청중을형간염표면항원(HBsAg)화을형간염e항원(HBeAg)농도적변화;형광정량PCR검측혈청화간조직중HBV DNA고패수;면역조직화학법검측간조직중HBV항원특이성;소목소-이홍(HE)염색관찰간조직병리변화.사용SPSS 17.0대수거간상관성진행선성상관분석. 결과 소서재주사cccDNA후,종제1천개시우혈액중검측도HBsAg화HBeAg적표체,량자균우제1주내신속승고체도최고치,병지속지제10주;기중HBsAg적표체수평경력료량개선상승재하강적과정(제4주위교저수평,제8주위교고수평),이HBeAg적표체수평경력료삼개선상승재하강적과정(제2주、제4주위교저수평,제3주、제6주위교고수평);형광정량PCR결과표명,10주후간조직중HBV DNA적고패수수착고、중、저제량조이체감,매극간조직중HBV DNA고패수[(1.14E+07)±(6.51E+06)、(9.81E+06)±(9.32E+06)、(3.72E+06)±(2.35E+ 06)]여주사적cccDNA농도(1.5、1.0、0.5 μg/ml)정정상관(피이삼상관계수r=0.979).10주후혈청중HBV DNA적고패수위중제량조최고,기차위고제량조,동시,각조혈청중적HBV DNA고패수교간조직중적저;면역조직화학결과현시,실험조라소서간조직중존재HBsAg화HBcAg적표체,HE염색결과현시간조직지방양혹공포양변성엄중,간소협조직결구불명현. 결론 본연구이용HBVcccDNA질립체내전도라소서,성공건립료HBV재간조직은정복제병지속표체HBV항원적라소서모형,위진일보연구HBVcccDNA만성지속감염적궤제화항병독치료타하기출.
Objective To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA.Methods Nude mice were injected with HBV cccDNA at doses of 1.5,1.0 or 0.5 μg/ml.A control group was generated by giving equal injection volumes of physiological saline.The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3,weeks 1-6,8 and 10 were assayed by reflection immunoassay.At post-injection week 10,all animals were sacrificed and liver tissues were collected.Copies of HBV DNA in serum and liver tissue were detected by real-time PCR.HBV antigens in liver tissue were detected of by immunohistochemistry.Pathological analysis of liver tissue carried out with hematoxylin-eosin staining.Linear correlation of data was determined by statistical analysis.Results HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10.The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8.In contrast,the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6.HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 μg/ml:1.14E+07 ± 6.51E+06 copies/g,1.0 μg/ml:9.81E+06 ± 9.32E+06 copies/g,and 0.5 μg/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979).HBV DNA copies in sera showed the pattern of 1.0 μg/ml cccDNA ＞ 1.5 μg/ml cccDNA ＞ 0.5 μg/ml cccDNA,and in general were higher than those detected in the liver tissues.Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg.HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice).Conclusion The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.