| 763 | 25 | 24 |
| 下载次数 | 被引频次 | 阅读次数 |
建立基于高压水动力法的乙型肝炎病毒(HBV)转染小鼠模型,并进一步建立和优化乙肝动物模型研究方法。首先构建了含腺相关病毒倒转末端重复序列元件与包含1.3个拷贝HBV基因组(ayw亚型)的HBV表达质粒(pAAV-HBV1.3);并将pAAV-HBV1.3质粒经高压水动力法尾静脉注射C57BL/6小鼠,不同时间点采集血液和肝组织标本,ELISA检测血清HBsAg、HBeAg表达;Real-time PCR检测血清及肝组织病毒载量;HE染色、免疫组化染色检测肝组织病理学改变及病毒抗原在肝组织中的定位及表达;最后采用免疫抑制剂地塞米松注射液(DEX)腹腔注射小鼠,建立免疫功能抑制小鼠模型,在此基础上制备乙肝病毒转染小鼠模型,并进行血清HBsAg、HBeAg检测。结果是正常免疫状态下,小鼠转染pAAV-HBV1.3 10d时血清及肝组织HBV相关抗原阳性,30d后HBV相关抗原检测阴性,但30d和60d血清及肝组织病毒载量检测均为阳性,且与对照组差异显著(P<0.01,P<0.05);经地塞米松注射后处于免疫抑制状态下的高压水动力法建立的乙肝病毒转染小鼠,则在60d仍可检测到HBsAg、HBeAg的表达。以上结果表明通过高压水动力法建立了急性乙肝小鼠模型,通过抑制小鼠免疫状态,可延长病毒在小鼠体内存留时间。该模型建立为HBV疫苗评价、药物开发及乙肝相关致病机理研究奠定了基础。
Abstract：To develop a HBV infection mouse model by hydrodynamic-based transfection and further to optimize the method of development of HBV infection mouse model.We first developed a construct which contained inverted terminal repeat elements(ITR) of adeno-associated virus(AAV) and 1.3 copies of HBV genome(ayw subtype).The pAAV-HBV1.3 DNA was then injected hydrodynamically into the tail veins of C57BL/6 mice in 5 seconds.The virus load in serum and liver was assayed by ELISA and Real-time PCR.The expression of virus antigen and the pathologic changes of liver were analyzed by HE and immunohistochemical staining.Meanwhile,to develop HBV transfected immunosuppressied mouse,mice were injected intraperitoneally triple with 0.2ml dexamethason(50mg/kg) every two days before HBV transfection.The levels of HBsAg and HBeAg were assayed by ELISA.Our data showed: ①HBsAg and HBeAg were positive(100%) in serum and liver of experimental normal mouse at day 10 after HBV transfection,and became negative at day 30 and day 60.Meanwhile the viral load in serum and liver in experimental group was significantly higher than that in control group at day 10,30 and 60 after HBV transfection(P<0.01,P<0.05,respectively).②HBsAg and HBeAg in serum in immunosuppressed mouse model were positive until 60 days.In conclusion,a HBV infection mouse model was developed successfully by hydrodynamic-based transfection.By suppressing the immune status of mice injected with dexamethasone,the expression of HBV antigens was extended longer than that in normal adult mice.These models pave a way for HBV research and evaluation of HBV vaccine and drug development.
[1]Blumberg B S.Hepatitis B virus,the vaccine,and thecontrol of primary cancer of the liver[J].Proc Natl AcadSci USA,1997,94:7121-7125.
[2]Liu W C,Mizokami M,Buti M,et al.Simultaneousquantification and genotyping of hepatitis B virus for gen-otypes A to G by Real-time PCR and two-step meltingcurve analysis[J].J Clin Microbiol,2006,44(12):4491-4497.
[3]Dandri M,Burda M R,T r k,et al.Repopulation ofmouse liver with human hepatocytes andin vivoinfectionwith hepatitis B virus[J].Hepatology,2001,4:981-988.
[4]Dandri M,Volz T K,Lütgehetmann M,et al.Animalmodels for the study of HBV replication and its variants[J].J Clin Virol,2005,34(1):s54-s62.
[5]Takehara T,Suzuki T,Ohkawa K,et al.Viral cova-lently closed circular DNA in a non-transgenic mousemodel for chronic hepatitis B virus replication[J].J Hep-atol,2006,44:267-274.
[6]Hiraga N,Imamura M,Tsuge M,et al.Infection ofHuman Hepatocyte Chimeric Mouse with Genetically En-gineered Hepatitis C Virus and its Susceptibility to Infec-tion[J].FEBS,2007,4:1983-1987.
[7]应若素,范学工,李宁,等.流体动力学法乙型肝炎病毒感染动物模型的建立[J].世界华人消化杂志,2006,14(11):1052-1057.
[8]Huang L R,Wu H L,Chen P J,et al.An immunocom-petent mouse for the tolerance of human chronic hepatitisB virus infection[J].Proc Natl Acad Sci USA,2006,103(47):17862-17867.
[9]Yang P L,Althage A,Chung J,et al.Hydrodynamicinjection of virual DNA:A mouse model of acute hepati-tis B virus infection[J].Proc Natl Acad Sci USA,2002,9(21):13825-13830.
[10]Liu F J,Liu L,He F,et al.Establishment and primaryapplication of a mouse model with hepatitis B virus rep-lication[J].World J Gastroenterol,2007,13(40):5324-5330.
[11]Konishi M,Tanaka H,Kaito M,et al.Ultrastructuraldemonstration of hepatitis B virus production in a mousemodel produced by hydrodynamic transfection[J].Int JMol Med,2007,20:31-36.
[12]Chen Z Y,He C Y,Meuse L,et al.Silencing of episo-mal transgene expression by plasmid bacterial DNA ele-mentsin vivo[J].Gene Ther,2004,11:856-864.
[13]Riu E,Grimm D,Huang Z,et al.Increased mainte-nance and persistence of transgenes by excision of ex-pression cassettes from plasmid sequencesin vivo[J].Human Gene Ther,2005,16:558-570.
[14]Liu F,Song Y K,Liu D.Hydrodynamics-based trans-fection in animals by systemic administration of plasmidDNA[J].Gene Ther,1999,6:1258-1266.
[15]Rehermann B,Nascimbeni M.Immunology of hepatitisB virus and hepatitis C virus infection[J].Nature,2005,5:215-229.
[16]李香兰,冯鹰.地塞米松对小鼠巨嗜细胞吞噬功能的影响[J].国际医药卫生导报,2005,11(20):6-7.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002060
中图分类号:R512.62
引用信息:
[1]郭燕菊,王文,孙世惠,等.免疫抑制剂地塞米松可明显延长乙型肝炎病毒抗原在水动力法转染HBV小鼠模型中的表达[J].病毒学报,2010,26(01):20-26.DOI:10.13242/j.cnki.bingduxuebao.002060.
基金信息:
艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2009ZX10004-401);; 重大新药创制科技重大专项(2009ZX09102-237)
2010-01-15
2010-01-15