中国医师杂志
中國醫師雜誌
중국의사잡지
Journal of Chinese Physician
2015年
10期
1509-1512
,共4页
瞬时受体电位通道/生物合成/药理学%RNA干扰%膀胱肿瘤/治疗/代谢/病理学%细胞增殖/药物作用%细胞凋亡/药物作用
瞬時受體電位通道/生物閤成/藥理學%RNA榦擾%膀胱腫瘤/治療/代謝/病理學%細胞增殖/藥物作用%細胞凋亡/藥物作用
순시수체전위통도/생물합성/약이학%RNA간우%방광종류/치료/대사/병이학%세포증식/약물작용%세포조망/약물작용
Transient receptor potential channels/BI/PD%RNA interference%Urinary bladder neoplasms/TH/ME/PP%Cell proliferation/DE%Apoptosis/DE
目的 探讨瞬时受体电位M8(TRPM8)对人膀胱癌细胞T24细胞增殖和凋亡的影响.方法 利用荧光定量PCR检测人膀胱移行细胞癌细胞MGH-U1、T24、BIU-87以及EJ中TRPM8的表达水平;设计并合成靶向TRPM8的特异性shRNA,通过脂质体转染法转染TRPM8表达最高的人膀胱癌细胞T24以构建稳定低表达TRPM8细胞株,通过免疫印迹法和荧光定量PCR来验证shRNA的干扰效率;通过四甲基偶氮唑蓝(MTT)比色法检测干扰后细胞的增殖能力;通过流式细胞仪检测细胞凋亡情况;免疫印迹法检测PI3K、细胞周期素DI(Cyclin D1)以及Bcl-2的表达水平.结果 T24的TRPM8表达量在4株膀胱移行细胞癌细胞中最高;特异性靶向TRPM8的shRNA能够有效下调T24细胞中TRPM8的表达;特异性shRNA干扰组细胞较其他两组细胞增殖显著减慢(F=64.73,P＜0.01);此外,干扰组的凋亡率显著高于空白组和对照组(F =84.73,P＜0.01);最后,TRPM8干扰组细胞的PI3K、Cyclin D1以及Bcl-2蛋白的表达较空白组和对照组显著下调.结论 采用RNA干扰技术能够有效沉默T24细胞的TRPM8基因,并致使其细胞增殖能力下降以及细胞凋亡率升高,其中可能的机制为通过调节细胞因子PI3K来调控人膀胱移行细胞癌细胞的增殖和凋亡.TRPM8在膀胱癌的发生发展中起着较为重要作用,TRPM8可能成为治疗膀胱癌的新靶点.
目的 探討瞬時受體電位M8(TRPM8)對人膀胱癌細胞T24細胞增殖和凋亡的影響.方法 利用熒光定量PCR檢測人膀胱移行細胞癌細胞MGH-U1、T24、BIU-87以及EJ中TRPM8的錶達水平;設計併閤成靶嚮TRPM8的特異性shRNA,通過脂質體轉染法轉染TRPM8錶達最高的人膀胱癌細胞T24以構建穩定低錶達TRPM8細胞株,通過免疫印跡法和熒光定量PCR來驗證shRNA的榦擾效率;通過四甲基偶氮唑藍(MTT)比色法檢測榦擾後細胞的增殖能力;通過流式細胞儀檢測細胞凋亡情況;免疫印跡法檢測PI3K、細胞週期素DI(Cyclin D1)以及Bcl-2的錶達水平.結果 T24的TRPM8錶達量在4株膀胱移行細胞癌細胞中最高;特異性靶嚮TRPM8的shRNA能夠有效下調T24細胞中TRPM8的錶達;特異性shRNA榦擾組細胞較其他兩組細胞增殖顯著減慢(F=64.73,P＜0.01);此外,榦擾組的凋亡率顯著高于空白組和對照組(F =84.73,P＜0.01);最後,TRPM8榦擾組細胞的PI3K、Cyclin D1以及Bcl-2蛋白的錶達較空白組和對照組顯著下調.結論 採用RNA榦擾技術能夠有效沉默T24細胞的TRPM8基因,併緻使其細胞增殖能力下降以及細胞凋亡率升高,其中可能的機製為通過調節細胞因子PI3K來調控人膀胱移行細胞癌細胞的增殖和凋亡.TRPM8在膀胱癌的髮生髮展中起著較為重要作用,TRPM8可能成為治療膀胱癌的新靶點.
목적 탐토순시수체전위M8(TRPM8)대인방광암세포T24세포증식화조망적영향.방법 이용형광정량PCR검측인방광이행세포암세포MGH-U1、T24、BIU-87이급EJ중TRPM8적표체수평;설계병합성파향TRPM8적특이성shRNA,통과지질체전염법전염TRPM8표체최고적인방광암세포T24이구건은정저표체TRPM8세포주,통과면역인적법화형광정량PCR래험증shRNA적간우효솔;통과사갑기우담서람(MTT)비색법검측간우후세포적증식능력;통과류식세포의검측세포조망정황;면역인적법검측PI3K、세포주기소DI(Cyclin D1)이급Bcl-2적표체수평.결과 T24적TRPM8표체량재4주방광이행세포암세포중최고;특이성파향TRPM8적shRNA능구유효하조T24세포중TRPM8적표체;특이성shRNA간우조세포교기타량조세포증식현저감만(F=64.73,P＜0.01);차외,간우조적조망솔현저고우공백조화대조조(F =84.73,P＜0.01);최후,TRPM8간우조세포적PI3K、Cyclin D1이급Bcl-2단백적표체교공백조화대조조현저하조.결론 채용RNA간우기술능구유효침묵T24세포적TRPM8기인,병치사기세포증식능력하강이급세포조망솔승고,기중가능적궤제위통과조절세포인자PI3K래조공인방광이행세포암세포적증식화조망.TRPM8재방광암적발생발전중기착교위중요작용,TRPM8가능성위치료방광암적신파점.
Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry.Expression of extracellular regulated kinase (ERK), cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P ＜0.01).Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P ＜ 0.01).In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.
