体内转导HBVcccDNA法建立HBV慢性感染的小鼠模型  

Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA

作　　者：赵婷婷[1] 李小松[2] 殷文伟[3] 蔡雪飞[2] 张文露[2] 陈飞兰[1] 赖国旗[1] 黄爱龙[2] 

机构地区：[1]重庆医科大学实验动物中心,400016 [2]重庆医科大学感染性疾病分子生物学教育部重点实验室 [3]重庆医科大学病毒性肝炎研究所

出　　处：《中华肝脏病杂志》2014年第4期260-265,共6页Chinese Journal of Hepatology

基　　金：重庆市科委科技创新能力建设项目（2010CB5013）

摘　　要：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HBVcccDNA以3μg／只、2μg／只、1ug／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HBVDNA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SPSS17．0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HBVDNA的拷贝数随着高、中、低剂量组而递减，每克肝组织中HBVDNA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、0．5μg／m1）呈正相关（皮尔森相关系数，=0．979）。10周后血清中HBVDNA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HBVDNA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, 1.0μg/ml： 9.81E＋06 ＋9.32E＋06 copies/g, and 0.5μg/ml： 3.72E＋06 ＋ 2.35E＋06 copies/g; Pearson＇sr = 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 show

关 键 词：肝炎病毒 乙型 DNA 共价闭合环状 水动力注射 持续感染 

分 类 号：R512.62[医药卫生—内科学]