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BALB/c裸鼠[4-6周龄,体质量（20±3）g]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通过降低ANG的表达、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。
Objective To determine the effect of angiogenin（ANG） down-regulation on the proliferation and apoptosis in human bladder cancer T24 cells.Methods An ANG-targeting siRNA vector and a non-homologous negative control were designed to transfect human bladder cancer T24 cells with Lipofectamine 2000,and the positive clones were screened by G418 and then named as T24-siANG and T24-NC,respectively.T24 cells without transfection were named as control（T24 group）.The expression of ANG at mRNA and protein levels was detected by RT-PCR and Western blotting.Cell proliferative activity was determined by MTT assay.Cell apoptosis was evaluated by TUNEL kit,Hochest（33342） and flow cytometry.The expression of caspase-3,Bax and Bcl-2 was determined by Western blotting.The tumor cells of the 3groups of cells at 2 × 106 were respectively injected into the back of BALB /c nude mice（4 to 6 weekes,20 ±3 g） to establish the xenograft models.The changes of micro-blood vessels in tumor tissue and the expression of CD31 were detected by immunofluorescence and HE staining.Immunohistochemical assay was used to detect the expression of ANG,Bax,Bcl-2 and caspase-3 in the tumors.Results Enzyme digestion and DNA sequencing verified the vectors was correctly constructed.The ANG expression at mRNA and protein levels was significantly decreased in the T24-siANG group（P 0.05）,and the cell proliferative activity reduced markedly（P 0.05）.Flow cytometry showed that the apoptotic rate was obviously higher in the T24-siANG cells than in the T24-NC cells and the T24 cells（P 0.01）.The number of TUNEL-positive cells was significantly higher in the T24-siANG cells than in the T24 cells and the T24-NC cells.The results of Hochest demonstrated that T24-siANG cells appeared typical apoptotic morphology features such as chromatin condensation,nuclear fragmentation and brighter blue fluorescence.However,the control cells did not have such apoptotic characteristics.BALB /c nude mice injected with the T24-siANG cells showed lower density of tumor microvessel and lower CD31 expression than those with the control cells（P 0.01）.Immunohistochemical assay results proved that the expression of Bcl-2 was significantly decreased in the T24-siANG cells as compared to that in the T24 and T24-NC cells（P 0.01）,but the expression of caspase-3 and Bax was stably increased（P 0.05）.Conclusion The plasmid expressing ANG-targeting siRNA downregulates ANG expression and inhibits the proliferation of T24 cells significantly,and thus induces apoptosis in bladder cancer T24 cells by regulating apoptosis-related proteins caspase-3,Bax and Bcl-2.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第14期1460-1466,共7页
Journal of Third Military Medical University
基金
国家自然科学基金(81372203)~~
关键词
血管生成素
膀胱癌
增殖
凋亡
angiogenin
bladder cancer
proliferation
apoptosis
