摘要
目的探索p53基因质粒联合反义c-myc基因质粒治疗人膀胱癌的作用机理。方法将可在真核细胞表达P53蛋白的质粒pCEP4p53和表达反义c-myc基因的质粒pCEP4ASCMH,以转染剂FuGENE6为介导,同时转染到人膀胱癌T24细胞中,经DNA测序、PCR-异双聚体PAGE电泳、Southern印迹杂交、蛋白质印迹法、ELISA、Caspase-3活性检测等方法分析实验结果。结果 DNA测序结果表明,转染所用的p53基因和反义c-myc基因的序列与文献报道一致,没有突变或失活等异常;PCR-异双聚体PAGE电泳证明T24细胞的p53基因发生了点突变;Southern印迹杂交实验确证p53基因和反义c-myc基因经FuGENE6介导转入靶细胞;蛋白质印迹法方法检测野生型p53基因在靶细胞内表达P53蛋白;ELISA方法检测瘤细胞内c-myc基因表达产物的含量水平下降;Caspase-3活性检测结果表明其活性增加,表现为典型的凋亡特征。结论 p53基因质粒联合反义c-myc基因质粒转染人T24膀胱癌细胞能诱导典型的凋亡,为膀胱癌的基因治疗提供了新途径。
Objective To study therapeutic mechanism of combination of p53 gene plasmid and antisense c-myc gene plasmid on human bladder cancer.Methods The plasmid pCEP4p53 expressing human P53 protein and plasmid pCEP4ASCMH ex-pressing antisense c-myc gene were co-transfected into human bladder cancer cell T24 strain with FuGENE6.DNA sequencing, PCR-Heteroduplex PAGE eletrophoresis,Southern Blot analysis ,Western Blot analysis,ELISA and Caspase-3 Activity assay were used for result analysis.Result DNA sequencing showed p53 or antisense c-myc sequence for transfection were consist-ent with those reported in published papers,with no abnormalities such as base mutation or loss.PCR-Heteroduplex PAGE e-lectrophoresis identified a single base mutation in p53 sequence of T24 cells.The p53 gene and antisense c-myc gene were con-firmed for successful transfection into target cells with FuGENE6 by Southern Blot analysis.Expression of p53 in the target cell by wt-p53 gene was confirmed by Western Blot analysis.ELISA showed decreased expression of c-myc level in T24 cells;Caspase-3 activity of transfected cells increased significantly,which was a classical apoptosis feature.Conclusion The co-transfection of p53 gene plasmid and antisense c-myc gene plasmid can induce classical apoptosis of Human Bladder Cancer Cell T24,which provided a new route for gene therapy of human bladder cancer.
出处
《江苏预防医学》
CAS
2015年第3期7-11,共5页
Jiangsu Journal of Preventive Medicine
基金
武汉轻工大学博士基金资助项目(142845)
