【作者】 张英元；

【导师】 何建行；

【作者基本信息】 广州医学院 ， 外科学， 2011， 硕士

【摘要】 目的:研究CEA启动子驱动的HSV-TK/CD双自杀基因系统在裸鼠体内的抑瘤作用及安全性。方法:使用普通RT-PCR验证SPC-A-1、SPC-A-1/TK+CD及SPC-A-1/pIRES-EGFP细胞的基因表达情况,然后建立裸鼠肺癌模型并进行分组:随机选用30只裸鼠,平均分为6组进行实验,分别为空白对照组、PBS对照组、空载体对照组、GCV治疗组、5-FC治疗组及双药治疗组。当肿瘤平均直径约为5mm时,开始治疗实验。PBS、GCV及5-FC经腹腔分别注入相应裸鼠体内,治疗5周后通过肿瘤平均体积、重量及生长抑制率评估抑瘤作用,进一步采用TUNEL法及透射电镜分析肿瘤细胞的凋亡情况,并通过常规病理切片HE染色观察心、肝、肾组织的病理改变。结果:普通RT-PCR证实SPC-A-1、SPC-A-1/TK+CD及SPC-A-1/pIRES-EGFP细胞皆为CEA阳性细胞,外源性双自杀基因HSV-TK/CD仅在SPC-A-1/TK+CD细胞中表达;在化疗前药GCV和(或)5-FC的作用下,双自杀基因HSV-TK/CD能对CEA表达阳性的肺癌细胞皮下移植瘤产生显著的抑制作用,通过TUNEL凋亡染色检测及电镜观察,提示HSV-TK/CD双自杀基因系统主要通过诱导肿瘤细胞凋亡从而发挥抑瘤作用,光镜下对裸鼠各器官进行观察,未发现明显的毒副作用。结论:CEA启动子驱动的HSV-TK/CD双自杀基因,在化疗前体药物GCV和(或)5-FC的作用下,对裸鼠体内CEA阳性的肺癌移植瘤具有明显的抑制作用,能诱导肿瘤细胞发生凋亡,但对裸鼠的重要器官无明显毒副作用。更多还原

【Abstract】 Objective: To investigate the treatment efficiency of the double suicide gene therapy system on lung carcinoma and the security in vivo studies.Method: Use the method of reverse transcription PCR to confirm the expression of gene in SPC-A-1,SPC-A-1/TK+CD and SPC-A-1/pIRES-EGFP cells.The total of 30 mice were randomly divided into 6 groups then established the model of human lung carcinoma xenografts, including blank control group, PBS control group, empty vector control group, GCV treatment group ,5-FC treatment group, combined treatment group..After tumors reached 5mm in diameter,the study began.Nude mice in Groups were given peritoneal injection of PBS、GCV、5-FC, respectively. After 5 weeks, the antitumor efficacy was evaluated by tumor mean volume, weight and inhibition rate .Moreover,we evaluated cell apoptosis by TUNEL and transmission electron microscope(TEM),and used routine pathologic examination(H.E)to observe the tissue of heart, liver, kidney.Result:The reverse transcription PCR proved SPC-A-1,SPC-A-1/TK+CD and SPC-A-1/pIRES-EGFP cells are CEA positive cells. exogenous double suicide gene HSV-TK/CD are only expressed by SPC-A-1/TK+CD cells, which combined with prodrug GCV and (or) 5-FC can significantly inhibite CEA positive lung carcinoma xenografts. Cell apoptosis were evaluated by TUNEL technique and transmission electron microscope (TEM),suggesting the anti-tumor effect of HSV-TK/CD double suicide gene system is through leading tumor cells to apoptosis. No obvious side effects are found in the tissue of various organs in nude mice by routine pathologic examination.Conclusion: To investigate the security and the treatment efficiency of the double suicide gene therapy system in a mouse xenografts model of lung carcinoma,we confirmed the HSV-TK/CD double suicide gene combined with prodrug GCV and (or) 5-FC can significantly inhibite CEA positive lung carcinoma cells through inducing targeted cells to apoptosis. Meanwhile, there are no significant side effects in other organs of nude mice.更多还原