TRPV2对膀胱肿瘤细胞生物学影响的实验研究

Study of biological effects of TRPV2 on bladder tumor cells

目的探讨TRPV2离子通道对膀胱肿瘤细胞株5637和EJ细胞增殖及迁移侵袭的影响。方法将人膀胱癌EJ细胞株分为空白组、对照组和sh-RNA干扰组;5637细胞株分为空白组,对照组和TRPV2转染组进行实验。经细胞筛选出单克隆阳性细胞后,采用Western blot检测TRPV2蛋白表达的变化,鉴定基因的转染效率。流式细胞仪检测细胞生长周期,MTT法检测细胞生长曲线,划痕法检测细胞迁移侵袭能力,Transwell法检测细胞的浸润能力。结果 Western blot结果显示EJ细胞干扰组的TRPV2蛋白表达量明显低于空白组与对照组;EJ细胞干扰组G1期细胞比例,显著高于空白组和对照组,差异有统计学意义(P<0.05);MTT法检测绘制细胞周期结果,3组细胞数目在第3天开始出现差异,干扰组的细胞数目显著低于空白组和对照组的细胞数(P<0.05),空白组与对照组细胞相比细胞周期无显著差异;划痕实验检测发现,干扰EJ细胞中的TRPV2表达后可以抑制EJ细胞的迁移;Transwell细胞浸润实验,说明干扰EJ细胞中TRPV2的表达后可以抑制肿瘤细胞的局部浸润作用。Western blot结果显示,干扰组5637细胞的TRPV2蛋白表达水平明显高于空白组和对照组;但3组细胞G1期细胞比例的差异无统计学意义;MTT法检测绘制细胞周期结果,3组细胞的生长曲线无明显差异;划痕实验检测发现5637细胞中TRPV2的表达量上升后可以明显加强5637细胞的迁移作用;Transwell浸润实验说明经过导入外源性的TRPV2使5637细胞中TRPV2的表达量上升后可以明显加强5637细胞的对于局部组织的浸润作用。结论 TRPV2可能是治疗膀胱肿瘤的关键靶点。

Objective To investigate the effect of cationic channel TRPV2 on proliferation,migration and invasion of bladder cancer 5637 and EJ cells. Methods TRPV2 over-expression plasmid was transfected into 5637 cells,and a sh-RNA-TRPV2 was transfected into EJ cells. The bladder tumor cells lines of EJ and bladder tumor cells lines of 5637 were divided into three groups: blank group,control group,interv or over-expression group; RTPCR and Western blot were used to assay the expression of the transfected plasmid. Cell proliferation abilities were assayed by flow cytometry( FCM) and MTT assay. Cell migration abilities were assayed by the scratch assay and transwell assay. Results Western blot result showed that the TRPV2 protein expression of EJ cell interference group was significantly lower than blank group and control group; EJ cells proportion of G1 phase in interference group was significantly higher than that of blank group and control group( P < 0. 05); cell cycle were detected and drawed by MTT method,the differences of cell number in three groups began to emerged on the third day,the cell number of interference group was significantly lower than that of blank group and control group( P < 0. 05),but there were no significant difference between blank control group and negative control group in cell cycle; scratch test showed that the migration of EJ cells was inhibited after the lower expression of TRPV2; Transwell assay showed that the local infiltration effect of EJ cells was inhibited after the lower expression of TRPV2. Western blot result showed that the TRPV2 protein expression of 5637 cells in TRPV2 transfected group was significantly higher than blank group and control group; 5637 cells proportion of G1 phase in three groups has no significantly differences( P < 0. 05); cell cycle were detected and drawed by MTT method,the differences of cell number in three groups was no obviously differences; scratch test showed that the migration of 5637 cells was enhanced after the higher expression of TRPV2; Transwell assay showed that the local infiltration effect of 5637 cells was enhanced after the higher expression of TRPV2. Conclusion TRPV2 may be a new target for therapy of bladder tumors.