瞬时感受器电位香草酸受体3离子通道蛋白在膀胱癌细胞中的表达及其对细胞增殖迁移能力的影响

Expression of transient receptor potential cation channel subfamily V member 3 in SW780 bladder carcinoma cell lines and its effects on proliferation and migration abilities

目的 观察瞬时感受器电位香草酸受体3离子通道蛋白（TRPV3）表达对体外膀胱癌细胞株SW780的增殖凋亡和生物学功能的影响.方法 应用基因转染方法观察TRPV3基因表达对体外膀胱癌细胞SW780的抗增殖作用和生物学功能的影响;通过免疫组织化学法和实时定量聚合酶链反应（Real-time PCR）技术检测TRPV3 mRNA在SW780细胞中的表达水平,Real-time PCR测定转染后SW780细胞中Ras、Raf、蛋白激酶B（Akt）和细胞外信号调节激酶（ERK） mRNA水平表达变化,并通过噻唑蓝（MTT）法、划痕实验、苏木素-伊红（HE）染色、显微镜等观察比较转染TRPV3基因的SW780细胞的迁移能力、增殖凋亡和形态学改变.结果 Real-time PCR和免疫组织化学结果证实转染后SW780细胞中有TRPV3稳定表达;Real-time PCR结果显示：A组Ras、Raf和Akt mRNA吸光度值分别为0.87±0.17、0.46±0.11和0.92±0.25,B组分别为1.71 ±0.32、1.63±0.29和1.95 ±0.38,C组分别为1.59±0.30、1.51±0.25和1.80 ±0.31.转染后SW780细胞中Ras、Raf和Akt mRNA水平显著降低（P＜0.01）.MTT法结果显示,SW780细胞A组（10∶1）24 h和48 h的抑制率分别为（19.58±2.83）％和（46.23 ±3.99）％,A组（20∶1）分别为（44.64 ±5.27）％和（68.73±7.51）％,A组（40∶1）分别为（59.63±6.85）％和（79.52±7.93）％.B、C组SW780细胞在24 h和48 h时无明显凋亡.表明随着TRPV3与SW780细胞效靶比增加及作用时间延长,抑制率明显增强（P＜0.01）.划痕实验结果显示：TRPV3转染的SW780细胞迁移能力低于正常SW780细胞.TRPV3作用于膀胱癌细胞24h后,形态学观察膀胱癌细胞发生凋亡或坏死.结论 TRPV3基因转染对体外膀胱癌细胞SW780细胞具有抑制增殖和促进凋亡作用.

Objective To study the effects of transient receptor potential cation channel subfamily V member 3 （TRPV3） gene on proliferation,apoptosis and biological functions of SW780 bladder carcinoma cell lines.Methods Effects of application of gene expression detected TRPV3 gene transcription of bladder carcinoma cell lines in vitro antiproliferative effect of SW780 and biological function;by detecting the expression level of TRPV3 mRNA immunohistochemical method and real-time quantitative polymerase chain reaction（Real-time PCR） technology in SW780 cells,Real-time PCR was measured after transfection,SW780 cells Ras,Raf,protein kinase B （Akt） and extracellular signal-regulated kinase （ERK） mRNA expression changes,and through the methyl thiazol tetrazolium （MTT） method,the scratch test,hematoxylin and eosin （HE） staining,microscope observation and comparison of TRPV3 gene transfection on the migration ability of SW780 cell proliferation,apoptosis and morphological changes.To analyse by using SPSS 14.0 statistical software.Results Real-time PCR and immunohistochemistry confirmed SW780 cells before and after transfection of TRPV3 stable expression;Real-time PCR results showed that group A of Ras and Raf and Akt mRNA absorbance value were 0.87 ± 0.17,0.46 ± 0.11 and 0.92 ± 0.25,respectively,group B were 1.71 ±0.32,1.63 ±0.29 and 1.95 ±0.38,respectively,group C were 1.59 ±0.30,1.51 ±0.25 and 1.80 ±0.31,respectively.the transfected SW780 cells in Ras,Raf and Akt mRNA level were significantly decreased （P 〈 0.01）.MTT assay showed that The inhibition rates of SW780 cells group A （10∶1） in 24 h and 48 h were 19.58 ±2.83 and 46.23 ± 3.99,respectively,group A （20∶1） were 44.64 ± 5.27 and 68.73 ± 7.51,respectively,group A （40∶1） was 59.63 ± 6.85 and 79.52 ± 7.93.B,C group of SW780 cells in 24 h and 48 h no significant apoptosis.TRPV3 and SW780 cells with effector target ratio increasing and prolonging of time,the inhibitory rate was enhanced obviously （P 〈 0.01）.The scratch test showed that TRPV3 transfected SW780 cell migration ability is lower than the normal SW780 cells.The effect of TRPV3 on SW780 cells 24 h,morphological observation of SW780 cells apoptosis or necrosis.Conclusion Transfection of TRPV3 gene can inhibit proliferation and promote apoptosis effect on pituitary adenoma cells in vitro SW780 cells.