Ezrin shRNA联合HSP70对骨肉瘤细胞凋亡和增殖的影响

 目的 探讨Ezrin基因沉默联合热休克蛋白（heat shock protein, HSP）70诱导的免疫杀伤对骨肉瘤细胞增殖和凋亡的影响。方法 采用人成骨肉瘤MG63细胞系作为研究对象，将人HSP70和Ezrin-shRNA的DNA片段分别克隆至含CMV和pU6双启动子的表达载体pGFP-V-RS中构建含HSP70和Ezrin-shRNA的真核表达载体pGFP-V-RS-shRNA和pGFP-V-RS-shRNA-HSP70。重组质粒分别转染入细胞，筛选出稳定表达的细胞克隆。荧光显微镜观察细胞形态及转染效果；荧光定量RT-PCR及蛋白印迹western blot检测稳定转染细胞株Ezrin和HSP70基因及蛋白水平的变化；采用MTT、流式细胞术检测细胞增殖、凋亡能力变化，Western blot检测凋亡及周期相关蛋白表达量变化；MTT法检测HSP70刺激产生特异性细胞毒性T淋巴细胞（CTL）对靶肿瘤细胞的杀伤作用。结果 荧光图像及基因和蛋白表达分析证实，首次成功构建同时沉默Ezrin和过表达HSP70的特异性载体。较单纯沉默Ezrin，同时过表达HSP70会在一定程度上减弱沉默Ezrin蛋白促进细胞凋亡和抑制增殖的效果，但与对照细胞相比，M63细胞凋亡率仍明显上升，自11.01%±0.22%上升至24.28%±0.50%，增殖速度则自395.14%±2.24%减少至310.00%±2.83%。另外，Western blot检测发现Ezrin-shRNA可促进凋亡基因Bax的表达，相反可降低抗凋亡基因Bcl-2和细胞周期蛋白Cyclin D1的表达。而Ezrin-shRNA/HSP70组较Ezrin-shRNA组促Bax表达和降低Bcl-2、细胞周期蛋白Cyclin D1表达有所减弱，但较阴性对照组仍有明显效果。MG63细胞的CTL细胞毒杀伤效应显示各效靶浓度下CT+IL-2+HSP70组的杀伤活性高于CT+IL-2组，最高达56.33%±1.95%。结论 同时沉默Ezrin、过表达HSP70既可以促进骨肉瘤细胞凋亡抑制其增殖，并利用HSP70诱导的CTL，增强对靶肿瘤细胞的杀伤作用。

Effects of Ezrin and heat shock protein 70 on apoptosis and proliferation of human osteosarcoma

Objective To investigate the influence of knocking down Ezrin expression in combination with HSP70-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells. Methods Human osteosarcoma cell line MG63 was cultured, the HSP70 and Ezrin-shRNA DNA fragments were cloned into the expression vector pGFP-V-RS containing CMV and pU6 promoters, and constructed the expression vector pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70. The vectors were transfected into MG63 cell line, respectively. The status of transfected MG63 cells was observed by fluorescent microscope. The expressions of Ezrin and HSP70 were examined by real time RT-PCR and Western blot. Flow cytometry、MTS test was used to detect the changes of cell apoptosis and proliferation, and changes of the expression of apoptosis and cell cycle related proteins was detected by Western blot. The specific cytotoxic T lymphocytes (CTLs) were induced by HSP70, and its killing effect on target MG63 tumor cells was analyzed by MTT assay. Results The specific vector simultaneously knocking down Ezrin and overexpressing HSP70 was constructed for the first time in China and confirmed by fluorescence microscope and gene/protein expression analysis. Compared to Ezrin knock-down alone, simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by Ezrin knock-down, however, when compared to the normal control, the apoptosis rate of LM8 cells was still significantly increased from 11.01%±0.22% to 24.28%±0.50%, while the proliferation rate decreased from 395.14%±2.24% to 310%±2.83%. In addition, Western detected that Ezrin-shRNA could promote the expression of Bax. However, the expression of Ezrin-shRNA could reduce the Bcl-2 and Cyclin D1. Ezrin-shRNA/HSP70 also has the same effect. MTT assays revealed that the CTL cytotoxic effect on target MG63 tumor cells at all concentrations were significantly higher in CT+IL-2+HSP70 group compared with that in CT+IL-2 group, with a cytotoxicity as high as 56.33%±1.95%. Conclusion Simultaneous knocking down Ezrin and overexpressing HSP70 promotes the apoptosis and inhibits the proliferation of osteosarcoma cell. And the HSP70 can induce CTL which enhances the killing effect on tumor cell. And the HSP70 can induce CTL which enhance the killing effect on tumor cell.