【摘要】 目的探讨转染小干扰RNA抑制膀胱尿路上皮癌T24细胞hTERT基因表达及对膀胱尿路上皮癌动物模型抑瘤率的影响,以探讨抑制hTERT基因实现抗肿瘤的作用,为膀胱尿路上皮癌的基因治疗提供新思路。方法以LipofectaminTM脂质体介导小干扰RNA转染膀胱尿路上皮癌T24细胞株,通过绘制细胞生长曲线、MTT比色法以及流式细胞仪检测观察小干扰RNA转染对T24细胞增殖的影响,PCR、Northern分析检测hTERT基因在T24细胞的表达,WesternBlot检测蛋白表达,T24细胞接种于裸鼠,分组给予抑制hTERT基因转染进行治疗,评价其抑瘤率。结果小干扰RNA抑制hTERT基因的表达可以抑制T24细胞的体外生长,实验组细胞的生长曲线较对照组明显降低;MTT比色显示实验组的细胞活力与对照组相比有显著性差异(P<0.05);PCR及Northern分析提示小干扰RNA抑制hTERT基因表达,WesternBlot检测到蛋白低表达;流式细胞仪检测显示基因转染使瘤细胞的生长滞留在G0+G1期,S期细胞明显减少,并观察到细胞凋亡。动物实验小干扰RNA抑制hTERT基因肿瘤抑制率可达34.4%,显著高于对照组(P<0.05),且两组间差异具有显著性(P=0.032)。结论小干扰RNA转染抑制hTERT基因表达对T24细胞具有体内外抗肿瘤的作用,提示小干扰RNA转染T24细胞抑制hTERT基因技术有可能为膀胱尿路上皮癌的基因治疗提供一种新的治疗策略与手段。更多还原

【Abstract】 【Objective】To investigate the inhibition proliferation effect of hTERT gene by small interfering RNA on human urothelium Carcinoma T24 cell lines in vitro and in vitro and the inhibitory rate in nude mice model so as to discuss hTERT gene transfection by small interfering RNA into T24 cell lines seeking a new way for gene therapy of carcinoma of human urothelium Carcinoma.【Methods】Small interfering RNA expression vector or pcDNA3.1(+) blank vector was transfected into human urothelium Carcinoma T24 cell lines and normal T24 cells were used as normal contro1.The inducing apoptosis effects of hTERT gene on cell growth was described by cell growth curve,MTT assay and flow cytometry(FCM) analysis.The expression of hTERT gene after transfection was detected by RT-PCR,WesternBlot assay for the protein.The nude mice model carried urothelium Carcinoma T24 cell lines were constructed,the nude mice were consecutively treated by hTERT gene transfection,the inhibitory tumor rate was estimated.【Results】Expression of hTERT gene strongly inhibited T24 cell proliferation in vitro.The growth of experimental group was significantly lower than that of contro1.MTT test showed that the difference of cell viability between experimental group and control cells was also significant(P <0.05).The expression of hTERT gene after transfection was high expression detected by RT-PCR,the high expression of protein was detected by Western Blot assay.FCM showed experimental group cells on S phase was reduced greatly and apoptosis could be observed in some cells.In nude mice of urothelium Carcinoma model,the inhibitory rate in hTERT gene transfection group got up to 34.4%(P <0.05),which differed remarkably(P =0.032).【Conclusions】The findings showed that the transfection of small interfering RNA to inhibit hTERT gene induced cellular apoptosis in T24 cells and that the urothelium Carcinoma of the nude mice model were inhibited by hTERT gene transfection which suggested that transfection of small interfering RNA to inhibit hTERT gene into T24 cells technique can be an effective means in the gene therapy of urothelium Carcinoma.更多还原