膀胱癌细胞Cx43对顺铂抗肿瘤作用影响研究

发布时间：2018-05-18 09:01

本文选题：膀胱癌 + Cx　；参考：《中华肿瘤防治杂志》2017年03期

【摘要】：目的连接蛋白43(connexin43,Cx43)在人体正常组织及肿瘤组织中广泛表达,并且参与细胞的生长控制和组织分化。本研究探讨采用RNA干扰技术沉默膀胱癌5637细胞株Cx43蛋白表达对顺铂化疗敏感性的影响。方法体外培养膀胱癌5637细胞和正常尿路上皮SV-HUC-1细胞,采用蛋白质印迹法检测膀胱癌5637细胞系和正常尿路上皮SV-HUC-1细胞中Cx43蛋白表达,应用免疫荧光技术检测膀胱癌5637细胞系中Cx43蛋白的定位。采用RNA干扰技术沉默膀胱肿瘤5637细胞株Cx43蛋白的表达并用顺铂(3μg/mL)处理SiRNA-Cx43组(实验组)和SiRNA-Control(对照组)后,通过CCK-8检测实验组、对照组及野生型5637细胞的增殖情况;采用流式细胞术检测两组癌细胞的凋亡;采用蛋白质印迹法检测顺铂处理后野生型5637细胞后细胞中Cx43、Cleaved Caspase-3的表达及实验组和对照组中Cx43、Cleaved Caspase-3和Bcl-2的表达。结果膀胱癌5637细胞中Cx43表达较正常尿路上皮细胞高,相对蛋白表达量分别为1.013±0.102和0.556±0.054,两细胞系比较差异有统计学意义,t=3.789,P=0.019;免疫荧光检测Cx43主要定位于细胞质中。CCK-8结果显示,膀胱癌5637细胞随着顺铂药物的浓度(0.75、1.5、3和6μg/mL)和时间(0、1、2、3d)的增加,细胞的增殖减少,采用重复测量方差分析,各组间比较,差异有统计学意义,F=153.634,P0.001;实验组增殖较对照组明显减少,差异有统计学意义,F=9.949,P=0.02。流式细胞仪检测结果显示,实验组和对照组凋亡率分别为(63.00±4.58)%和(34.33±6.03)%,差异有统计学意义,t=7.457,P0.01。蛋白质印迹法结果显示,5637细胞随着药物(顺铂3μg/mL)作用时间增加,Cx43蛋白表达逐渐降低,差异有统计学意义,F=178.868,P0.001;而Cleaved Caspase-3逐渐升高,差异有统计学意义,F=21.643,P0.001。顺铂(3μg/mL,4h)处理实验组和对照组后,Cleaved Caspase-3蛋白相对表达量分别为0.740±0.092和0.373±0.091,差异统计学意义,t=6.394,P=0.001;BclL-2蛋白相对表达量分别为0.260±0.066和0.817±0.068,差异统计学意义,t=4.814,P=0.005结论沉默膀胱癌5637细胞中Cx43蛋白表达能提高顺铂的敏感性,可能与反常定位于细胞质中的Cx43参与线粒体介导的凋亡途径有关。
[Abstract]:Objective connexin 43) is widely expressed in human normal tissues and tumor tissues, and is involved in cell growth control and tissue differentiation. The aim of this study was to investigate the effect of RNA interference technique on the chemosensitivity of cisplatin to bladder cancer cell line 5637 by silencing the expression of Cx43 protein. Methods bladder cancer cell line 5637 and normal urinary tract epithelial SV-HUC-1 cell line were cultured in vitro. The expression of Cx43 protein in 5637 cell line and SV-HUC-1 cell line of normal urinary tract epithelium was detected by Western blotting. The localization of Cx43 protein in 5637 cell line of bladder cancer was detected by immunofluorescence technique. The expression of Cx43 protein in 5637 cell line of bladder tumor was silenced by RNA interference technique and treated with cisplatin 3 渭 g / mL. The proliferation of SiRNA-Cx43 group (experimental group) and SiRNA-Controll group (control group) were detected by CCK-8. Apoptosis of two groups of cancer cells was detected by flow cytometry, the expression of Cx43 Caspase-3 in wild type 5637 cells treated with cisplatin and the expression of Cx43 Caspase-3 and Bcl-2 in the experimental group and control group were detected by Western blot. Results the expression of Cx43 in 5637 cells was higher than that in normal urothelial cells (1.013 卤0.102 and 0.556 卤0.054, respectively), and the difference between the two cell lines was statistically significant. The results of immunofluorescence assay showed that the expression of Cx43 was mainly located in the cytoplasm of .CCK-8. The proliferation of bladder cancer 5637 cells decreased with the increase of cisplatin concentration of 0.75 渭 g / mL and 6 渭 g / mL, and time of 2d. By repeated measurement of variance, the difference was statistically significant between the two groups, and the proliferation of the experimental group was significantly lower than that of the control group. The difference was statistically significant (P < 0.05). The results of flow cytometry showed that the apoptotic rates of the experimental group and the control group were 63.00 卤4.58% and 34.33 卤6.03%, respectively. The difference was statistically significant (P 0.01). The results of Western blotting showed that the expression of Cx43 protein decreased with the increase of Cisplatin (Cisplatin 3 渭 g / mL) time, and the difference was statistically significant (P 0.001), while the Cleaved Caspase-3 increased gradually, the difference was statistically significant (P 0.001). The relative expression of Cleaved Caspase-3 protein was 0.740 卤0.092 and 0.373 卤0.091, respectively, and the relative expression of BclL-2 protein was 0.260 卤0.066 and 0.817 卤0.068 respectively in 5637 cells with silencing bladder cancer. White expression can increase the sensitivity of cisplatin. It may be related to the involvement of Cx43 in mitochondria mediated apoptosis.
【作者单位】：承德医学院研究生学院;承德医学院附属医院泌尿外科;
【分类号】：R737.14

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