本文選題：膀胱癌 + Cx��； 參考：《中華腫瘤防治雜志》2017年03期

【摘要】：目的連接蛋白43(connexin43,Cx43)在人體正常組織及腫瘤組織中廣泛表達(dá),并且參與細(xì)胞的生長控制和組織分化。本研究探討采用RNA干擾技術(shù)沉默膀胱癌5637細(xì)胞株Cx43蛋白表達(dá)對順鉑化療敏感性的影響。方法體外培養(yǎng)膀胱癌5637細(xì)胞和正常尿路上皮SV-HUC-1細(xì)胞,采用蛋白質(zhì)印跡法檢測膀胱癌5637細(xì)胞系和正常尿路上皮SV-HUC-1細(xì)胞中Cx43蛋白表達(dá),應(yīng)用免疫熒光技術(shù)檢測膀胱癌5637細(xì)胞系中Cx43蛋白的定位。采用RNA干擾技術(shù)沉默膀胱腫瘤5637細(xì)胞株Cx43蛋白的表達(dá)并用順鉑(3μg/mL)處理SiRNA-Cx43組(實(shí)驗(yàn)組)和SiRNA-Control(對照組)后,通過CCK-8檢測實(shí)驗(yàn)組、對照組及野生型5637細(xì)胞的增殖情況;采用流式細(xì)胞術(shù)檢測兩組癌細(xì)胞的凋亡;采用蛋白質(zhì)印跡法檢測順鉑處理后野生型5637細(xì)胞后細(xì)胞中Cx43、Cleaved Caspase-3的表達(dá)及實(shí)驗(yàn)組和對照組中Cx43、Cleaved Caspase-3和Bcl-2的表達(dá)。結(jié)果膀胱癌5637細(xì)胞中Cx43表達(dá)較正常尿路上皮細(xì)胞高,相對蛋白表達(dá)量分別為1.013±0.102和0.556±0.054,兩細(xì)胞系比較差異有統(tǒng)計(jì)學(xué)意義,t=3.789,P=0.019;免疫熒光檢測Cx43主要定位于細(xì)胞質(zhì)中。CCK-8結(jié)果顯示,膀胱癌5637細(xì)胞隨著順鉑藥物的濃度(0.75、1.5、3和6μg/mL)和時(shí)間(0、1、2、3d)的增加,細(xì)胞的增殖減少,采用重復(fù)測量方差分析,各組間比較,差異有統(tǒng)計(jì)學(xué)意義,F=153.634,P0.001;實(shí)驗(yàn)組增殖較對照組明顯減少,差異有統(tǒng)計(jì)學(xué)意義,F=9.949,P=0.02。流式細(xì)胞儀檢測結(jié)果顯示,實(shí)驗(yàn)組和對照組凋亡率分別為(63.00±4.58)%和(34.33±6.03)%,差異有統(tǒng)計(jì)學(xué)意義,t=7.457,P0.01。蛋白質(zhì)印跡法結(jié)果顯示,5637細(xì)胞隨著藥物(順鉑3μg/mL)作用時(shí)間增加,Cx43蛋白表達(dá)逐漸降低,差異有統(tǒng)計(jì)學(xué)意義,F=178.868,P0.001;而Cleaved Caspase-3逐漸升高,差異有統(tǒng)計(jì)學(xué)意義,F=21.643,P0.001。順鉑(3μg/mL,4h)處理實(shí)驗(yàn)組和對照組后,Cleaved Caspase-3蛋白相對表達(dá)量分別為0.740±0.092和0.373±0.091,差異統(tǒng)計(jì)學(xué)意義,t=6.394,P=0.001;BclL-2蛋白相對表達(dá)量分別為0.260±0.066和0.817±0.068,差異統(tǒng)計(jì)學(xué)意義,t=4.814,P=0.005結(jié)論沉默膀胱癌5637細(xì)胞中Cx43蛋白表達(dá)能提高順鉑的敏感性,可能與反常定位于細(xì)胞質(zhì)中的Cx43參與線粒體介導(dǎo)的凋亡途徑有關(guān)。
[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 鹵0.102 and 0.556 鹵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 鹵4.58% and 34.33 鹵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 鹵0.092 and 0.373 鹵0.091, respectively, and the relative expression of BclL-2 protein was 0.260 鹵0.066 and 0.817 鹵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.
【作者單位】： 承德醫(yī)學(xué)院研究生學(xué)院;承德醫(yī)學(xué)院附屬醫(yī)院泌尿外科;
【分類號】：R737.14

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本文編號：1905241