siRNA沉默血管生成素对人膀胱癌T24细胞的调亡的影响.docVIP

下调血管生成素对人膀胱癌T24细胞增殖、凋亡的影响 舒 静，田文琳，李 林，刘玉林，陈俊霞 (400016 重庆，重庆医科大学基础医学院细胞生物学与遗传学教研室，分子医学与肿瘤研究中心) ［摘要］ 目的 构建人血管生成素(angiogenin，ANG)基因siRNA干扰质粒，检测其对膀胱癌T24细胞增殖、凋亡的影响。方法 将针对人ANG基因siRNA表达质粒和无同源性的对照质粒稳定转染T24细胞后经G418筛选出阳性克隆。根据转染质粒不同分为3组：T24组(未转染组)、T24-NC组(转染阴性对照质粒组)、T24-siANG组(转染干扰质粒组)。通过RT-PCR检测T24细胞中ANG的mRNA表达水平，Western blot检测ANG蛋白的表达；MTT法检测细胞增殖活力；流式细胞术、TUNEL及Hochest(33342)检测细胞凋亡；Western blot检测凋亡相关蛋白Bcl-2、Bax及Caspase-3的表达；构建移植瘤模型，组织HE染色和免疫荧光CD31检测微血管变化；组织免疫化学方法检测移植瘤中ANG、Bax、Caspase-3、Bcl-2的表达。结果 酶切和测序结果证明重组质粒构建成功；T24-siANG组ANG的mRNA水平及蛋白表达水平显著降低（P＜0.05）；T24-siANG组细胞增殖活力明显下降（P＜0.05）；流式细胞仪检测结果显示：与T24-NC组和T24细胞组相比，T24-siANG组细胞的凋亡率明显升高(P＜0.01)；TUNEL检测结果显示：T24-siANG组出现大量凋亡细胞；Hochest检测结果显示：T24-siANG组出现典型的凋亡形态特征如染色质凝集、核碎裂和明亮的蓝色荧光等；Western blot结果显示，在T24-siANG组中，Bcl-2的表达明显降低(P＜0.01)，而Bax和激活的Caspase-3的表达明显升高(P＜0.05)；组织HE染色和组织免疫荧光显示：T24-siANG组肿瘤微血管生成较T24与T24-NC组明显减少(P＜0.01)；组织免疫化学结果也表明T24-siANG组ANG、Bcl-2蛋白显著降低，而Bax、活化的Caspase-3蛋白明显升高。结论 成功构建的干扰质粒能有效降低ANG的表达，显著抑制膀胱癌T24细胞的增殖，调节凋亡关键蛋白的表达，从而促进膀胱癌T24细胞的凋亡。 ［关键词］ 血管生成素；膀胱癌；增殖；凋亡 [中图法分类号] [文献标志码] A Effect of down-regulation human angiogenin on proliferation and apoptosis in human bladder cancer T24 cells Shu Jing ,Tian Wenlin, Li Lin ,Liu Yulin ,Chen Junxia (Dapartment of Cell Biology and Genetics ,Molecular Medicine and Cancer Research Center ,College of Basic Medical Science ,Chongqing Medical University, Chongqing, 400016,China) ［Abstract］ Objective To determine the effects of down-regulation angiogenin on proliferation, apoptosis in human bladder cancer T24 cells. Methods One siRNA vectors specific to ANG gene and one non-homologous negative control were designed and constructed, then transfected into bladder cancer T24 cells with Lipofectamine 2000, and the positive clones were screened by G418, respectively, and were named as T24-siANG, T24-NC.T24 cells without transfection were named as control (T24 group). The expression of ANG gene at mRNA and protein levels was detected by RT-PCR,