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目的:为构建携带可调控自杀基因的重组腺相关病毒载体。方法:将含有Tet-On基因调节系统及自杀基因HSVtk的重组腺相关病毒载体pAAV/TRE/HSVtk/Tet-On,与辅助质粒pAAV-RC、pHelper以磷酸钙共沉淀的方法转染HEK293细胞,进行病毒包装后得到了AAV/TRE/HSVtk/Tet-On重组腺相关病毒,以氯化铯密度梯度离心的方法对包装好的病毒进行纯化。用纯化后的重组腺相关病毒感染乳腺癌细胞株MGF- 7,用MTT法及RT-PCR检测在Dox诱导下,GCV对rAAV感染的MCF-7细胞的杀伤作用以及HSvtK基因在MCF-7细胞内的表达情况。结果在rAAV+Dox+GCV组,GCV对rAAV感染(MOI=10~4、10~5、10~6)的MCF-7细胞的杀伤作用明显高于rAAV+Dox组,并且RT-PCR结果显示经Dox诱导HSVtk表达较明显。结论:成功构建了携带可调控自杀基因的重组腺相关病毒载体,该病毒载能有效的感染乳腺癌细胞MCF-7,并能联合GCV治疗,抑制肿瘤细胞生长,从而为腺相关病毒介导的可调控自杀基因治疗乳腺癌动物实验奠定了基础。
Objective: To construct recombinant adeno-associated virus vector carrying suicide gene. METHODS: HEK293 cells were transfected with the recombinant adeno-associated virus vector pAAV / TRE / HSVtk / Tet-On containing the regulatory system of Tet-On gene and the HSVtk suicide gene and the helper plasmids pAAV-RC and pHelper by calcium phosphate coprecipitation. After packaging the virus, AAV / TRE / HSVtk / Tet-On recombinant adeno-associated virus was obtained and the packaged virus was purified by cesium chloride density gradient centrifugation. The purified recombinant adeno-associated virus was used to infect MCF-7 breast cancer cell line MGF-7. MTT assay and RT-PCR were used to detect the killing effect of GCV on rAAV-infected MCF-7 cells and the effect of HSvtK on MCF-7 cells Within the expression. Results In the rAAV + Dox + GCV group, the cytotoxicity of GCV against rAAV infection (MOI = 10 ~ 4,10 ~ 5,10 ~ 6) was significantly higher than that of rAAV + Dox group After Dox induced HSVtk expression more obvious. CONCLUSION: Recombinant adeno-associated virus vector carrying suicide gene can be successfully constructed, which can effectively infect breast cancer cell MCF-7 and can be combined with GCV treatment to inhibit the growth of tumor cells and thus to be adeno-associated virus-mediated The regulation of suicide gene therapy for breast cancer animal experiments laid the foundation.