【作者】 马衣努尔﹒买提托合提；

【导师】 王世宣；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 硕士

【摘要】 第一部分(Part I)重组腺相关病毒-LIGHT对小鼠宫颈癌模型的治疗作用研究[摘要]目的构建含肿瘤坏死因子超家族成员14(LIGHT)的重组腺相关病毒(rAAV-LIGHT),探讨(rAAV-LIGHT)对TC-1细胞株所建立的小鼠宫颈癌模型的抑瘤效应的可行性。方法用基因重组方法构建含LIGHT全长的重组腺相关病毒骨架质粒pAAV-IRES-LIGHT-hrGFP,磷酸钙沉淀法将腺相关病毒载体系统共转染入病毒包装细胞中,分别合成rAAV-LIGHT和rAAV-GFP。并用实时荧光定量PCR测病毒滴度。RT-PCR检测rAAV-LIGHT对TC-1细胞的转染效率。体外用淋巴毒性试验来检测LIGHT的免疫效应。小鼠细胞系TC-1(转染过E6E7RAS)用于建立小鼠宫颈癌模型。成瘤第3天对空白对照组(5只小鼠),rAAV-GFP(5只小鼠), rAAV-LIGHT(5只小鼠)组分别进行肌肉注射。观察其生物特性及用实时定量PCR检测肿瘤微环境中LIGHT和其他细胞因子的表达情况。观察rAAV-LIGHT的抑瘤效应。通过流式细胞仪和肿瘤组织H&E染色、免疫组化检测小鼠淋巴细胞增值效应。结果成功构建并鉴定了rAAV-LIGHT和rAAV-GFP,滴度为2.5×1010 v.g/ml,转染效率为60%。SPSS13.0分析结果显示rAAV-LIGHT组肿瘤大小明显小于对照组及rAAV-GFP组(P=0.0055,P=0.029 P<0.05)。对照组及rAAV-GFP组比较无明显统计学意义(P=0.115 P>0.05)。结论rAAV-LIGHT可在小鼠宫颈癌模型逆转肿瘤生长。第二部分(Part 2)携带小鼠HVEM的重组腺相关病毒载体的构建及鉴定【摘要】目的克隆小鼠疱疹病毒侵入介体(HVEM)基因,构建其重组腺相关病毒载体,鉴定目的基因在真核细胞中表达,为免疫基因治疗奠定基础。方法用RT-PCR法从小鼠脾脏淋巴细胞克隆出全长HVEM基因,构建携带HVEM基因的穿梭质粒pAAV-IRES-HVEM-hrGFP和辅助质粒pAAV-RC,pHelper利用磷酸钙共沉淀法将穿梭质粒共转染入AAV-293细胞中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒rAAV- HVEM。用氯仿-PEG8000法纯化病毒,实时荧光定量PCR测病毒滴度。RT-PCR检测rAAV- HVEM在中国仓鼠卵巢(CHO)细胞中的表达情况。结果成功构建组装重组小鼠HVEM腺相关病毒,纯化后病毒滴度为5×1010 v.g/mL,且在CHO转导细胞中能有效表达目的基因。结论构建的重组腺相关病毒载体rAAV-HVEM为组织工程相关细胞转基因构建及临床应用提供先进的载体系统。为今后进一步研究HVEM的功能及其应用奠定基础。更多还原

【Abstract】 AAV mediated local delivery of LIGHT suppresses tumor-igenesis in a murine cervical cancer model[Abstract] Objective To explore the feasibility of reverse effect of recombinant LIGHT adeno-associated virus(rAAV-LIGHT) in mouse cervical cancer model. Methods Using DNA recombination technique to construct AAV main plasmid LIGHT. rAAV-LIGHT and rAAV-GFP were produced by co-transfaction efficiency was measured.The titer was measured by Quantitative Real-time PCR .After transfactin rAAV-LIGHT to TC-1 cells the espression of LIGHT was measured by RT-PCR. Using lymphocyte cytotoxcity assay to test the immunity effects of LIGHT. Mouse cell line TC-1 (transfected by E6E7RAS) was used in the establishment of the cervical cancer model. Three groups of cervical cancer models applied by PBS(as control)(5 mice), rAAV-GFP(5 mice), rAAV-LIGHT(5 mice) intramuscularly on the 3d after innoculation.The biologic feature of the model was observed and the espression of LIGHT and other cytokine experession in the tumor microenvironment was measured by Real-time PCR. Then the effect of rAAV-LIGHT on supperssion tumor growth in models were observed. Analyzed the mice spleen lymphocyte proliferation by flow cytometry, Tumor tissue H&E and Immunohistochemistry. Results rAAV-LIGHT and rAAV-GFP were constructed and identified successfully.The titers were both 2.5×1010 virus particles/ml. The transfection efficiency was 60%. SPSS13.0 analysis showed rAAV-LIGHT group tumor size smaller than control and rAAV-GFP(P=0.0055,P=0.029 P < 0.05). The control and rAAV-GFP group showed no statistically difference in tumor size(P=0.115 P>0.05). Conclusion rAAV-LIGHT could reverse tumor growth in cenrvical cancer model. Construction and identification of recombinant mouse HVEM adeno-associated virus【Abstract】Objective To clone mouse HVEM gene into AAV vector pAAV-IRES-hrGFP and obtain high titer and purity recombinant mouse HVEM-AAV and infection of CHO cells for expression, to lay the foundation for immuno-gene therapy. Methods Framework plasmid of recombinant HVEM-AAV was constructed by gene recombination. The recombinant plasmid pAAV-IRES-HVEM-hrGFP , pAAV-RC ,pHelper were co-transfected into AAV-293 cells according to the calcium phosphate based protocol and produced recombinant virus rAAV-HVEM purified by PEG/chloroform. The purity of recombinant rAAV-HVEM was verified by SDS-PAGE. The titer of the virus particles was detected by Quantitative Real-Time PCR. HVEM expression in CHO cells which were infected by recombinant rAAV-HVEM was verified by RT-PCR and FACS analysis. Results Recombinant mouse rAAV-HVEM was successfully constructed with high titer and high purity. After transfection recombinant AAV-HVEM mediated a stable expression of HVEM mRNA in CHO cells。Conclusion Recombinant mouse rAAV-HVEM is successfully constructed, which will benefit for further study the function of HVEM and its applications.更多还原