【作者】 陈道桢；

【导师】 张丽珊；

【作者基本信息】 东南大学 ， 遗传学， 2003， 硕士

【摘要】 目的 探讨逆转录病毒（RV）载体介导的单纯疱疹病毒胸苷激酶（HSV -TK）基因转染小鼠乳腺癌细胞，联合抗病毒药物丙氧鸟苷（GCV）对小鼠乳腺癌细胞系MA782/5S-8102体外及体内杀伤作用及其产生的旁观者效应。方法 采用脂质体转染法将GINaTK载体转入包装细胞PA317 ,PCR和扫描电镜检测HSV-TK基因整合和病毒颗粒分泌情况。取滴度最高的病毒上清液感染小鼠乳腺癌细胞 MA782/5S-8102，经G418筛选后，得到带有HSV-TK基因的MA782/5S-8102/TK细胞；然后将MA782/5S-8102/TK细胞分别用于体外和体内实验。 MTT法观察GCV对离体乳腺癌细胞的毒性作用和旁观者效应； 体内实验：分动物致瘤组 、抑瘤实验组 和治疗实验组 。分别按实验组别要求接种5.0 ×10~6细胞/只于 BALB/C小鼠的右腋窝皮下， 观察各组肿瘤形成及肿瘤治疗情况并统计各组生存期。治疗结束后将标本制成石蜡切片进行病理学分析,RT-PCR检测HSV-TK基因在肿瘤组织中的表达情况。统计学处理采用SPSS软件进行完全随机的方差分析（ANOVA）。结果 载体GINaTK导入了PA317细胞，阳性克隆命名为PA317/TK。 PCR检测 PA317/TK细胞出现一特异性404bp 阳性条带，而 PA317细胞没有扩增出相应条带 。PA317/TK细胞经扫描电镜检测见其表面有颗粒状突起，其周围有许多呈团状的病毒颗粒。病毒滴度为4.0×10~4 cfu/ml。用病毒上清液感染 MA782/5S-8102，成功得到了表达HSV -TK基因的MA782/5S-8102/TK细胞。体外实验结果显示，当MA782/5S-8102/TK细胞数占混合细胞10 %时，低浓度(10μg/ml)的GCV就可将50%左右的肿瘤细胞杀死。体内 实验结果显示小鼠成瘤率为100%。 GCV可明显抑制MA782/5S-8102/TK细胞在BALB/C小鼠体内的肿瘤形成。 经GCV治疗后，MA782/5S-8102/TK组和混合细胞组的肿瘤体积分别较对照MA782/5S-8102组肿瘤体积缩小约36.7%和28.6 % （均p<0.001）; 生存期也明显延长（p<0.001）；RT-PCR检测HSV-TK基因在肿瘤组织中有表达。实验组肿瘤组织与对照组相比存在明显的病理学改变。结论 逆转录病毒可介导HSV -TK基因转入小鼠乳腺癌细胞 MA782/5S-8102 并获稳定表达， HSV-TK/GCV自杀基因系统在体内外对乳腺癌细胞均有杀伤作用，且存在明显的旁观者效应。更多还原

【Abstract】 Objective:To study in vitro and in vivo killing effect and the bystander effect of retrovirus-mediated herpes simplex virus thymidine kinase (HSV-TK)gene transference following administration of ganciclovir (GCV) on mice breast carcinoma cells MA782/5S-8102. Methods: GINaTK retroviral vector containing HSV-TK gene was transduced into PA317 packaging cell by lipofectin, and positive clones(named PA317/TK) were respectively measured with PCR and with scan electron microscrope. Mice breast carcinoma cell line MA782/5S-8102 was infected by high titer viral supernatant. PCR Was resorted to demonstrate the successful transduction of the HSV-TK gene. MA782/5S-8102 /TK cells and MA782/5S-8102 cells were used in in vitro and in vivo study. In in vitro study, sensitivity of MA782/5S-8102 /TK cells to GCV and bystander effect were observed by MTT test. The killing effects and bystander effects of HSV-TK/GCV system on breast carcinoma cells were also in in vivo studied.40 female BALB/C 6-8 week old mice were dividided into three groups at random : tumors formation group, tumors inhibition group and tumors therapy group.Each mouse was implanted 5.0 ×10~6 MA782/5S-8102 or MA782/5S-8102 /TK cells or mixture cells in right axilla. At the end of the experiment,mice were sacrificed and the specimens were processed for histopathological analysis. RT-PCR was applied to detect the expression of HSV-TK gene in tumor tissure .Statistical analysis of the data was performed using the ANOVA(analysis of variance)by SPSS software. Results: PA317 cells were transfected successfully with HSV-TK gene by lipofectin(named PA317/TK). The titer is 4.0×10~4 cfu/ml. Experimental results revealed that stable virus producing cell line was established and MA782/5S-8102/TK cells expressing the HSV-TK gene were obtained successfully. In in vitro study , when the ratio of MA782/5S-8102 /TK cells reached 10%,the tumor cell-killing proportion was almost 50 %.In in vivo study, the ratio of tumors formation is<WP=6>100%.GCV could suppress tumor formation of the MA782/5S-8102/TK cells. After mice treated with GCV,the median tumor volume of mice implanted with MA782/5S-8102/TK cells and mice with mixture cells was respectively decreased to 63.3%( p<0.001) and 71.4%( p<0.001) compared with the control tumors.Their median survival was significantly prolonged（p<0.001）.Tumors treated with GCV revealed different histopathological features compared with the control tumors. The expression of HSV-TK gene was detected by RT-PCR.Conclusion:The test showed that the HSV-TK gene can be transducted into mice breast cancer line MA782/5S-8102 under the mediation of retrovirus and be stable expressed, HSV-TK/GCV suicide gene therapy system could improve the antitumoral efficiency . The bystander effect could be observated in HSV-TK/GCV system in in vitro and in vivo.更多还原