中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2015年
2期
165-173
,共9页
骨肉瘤%HSP70热休克蛋白质类%细胞凋亡%细胞增殖
骨肉瘤%HSP70熱休剋蛋白質類%細胞凋亡%細胞增殖
골육류%HSP70열휴극단백질류%세포조망%세포증식
Osteosarcoma%HSP70 heat-shock proteins%Apoptosis%Cell proliferation
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Eznn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-shRNMHSP70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.
目的 探討Ezrin基因沉默聯閤熱休剋蛋白(heat shock protein,HSP)70誘導的免疫殺傷對骨肉瘤細胞增殖和凋亡的影響.方法 採用人成骨肉瘤MG63細胞繫作為研究對象,將人HSP70和Ezrin-shRNA的DNA片段分彆剋隆至含CMV和pU6雙啟動子的錶達載體pGFP-V-RS中構建含HSP70和Ezrin-shRNA的真覈錶達載體pGFP-V-RS-shRNA和pGFP-V-RS-shRNA-HSP70.重組質粒分彆轉染入細胞,篩選齣穩定錶達的細胞剋隆.熒光顯微鏡觀察細胞形態及轉染效果;熒光定量RT-PCR及蛋白印跡western blot檢測穩定轉染細胞株Eznn和HSP70基因及蛋白水平的變化;採用MTT、流式細胞術檢測細胞增殖、凋亡能力變化,Western blot檢測凋亡及週期相關蛋白錶達量變化;MTT法檢測HSP70刺激產生特異性細胞毒性T淋巴細胞(CTL)對靶腫瘤細胞的殺傷作用.結果 熒光圖像及基因和蛋白錶達分析證實,首次成功構建同時沉默Ezrin和過錶達HSP70的特異性載體.較單純沉默Ezrin,同時過錶達HSP70會在一定程度上減弱沉默Ezrin蛋白促進細胞凋亡和抑製增殖的效果,但與對照細胞相比,M63細胞凋亡率仍明顯上升,自11.01％±0.22％上升至24.28％±0.50％,增殖速度則自395.14％±2.24％減少至310.00％±2.83％.另外,Western blot檢測髮現Ezrin-shRNA可促進凋亡基因Bax的錶達,相反可降低抗凋亡基因Bcl-2和細胞週期蛋白Cyclin D1的錶達.而Ezrin-shRNMHSP70組較Ezrin-shRNA組促Bax錶達和降低Bcl-2、細胞週期蛋白Cyclin D1錶達有所減弱,但較陰性對照組仍有明顯效果.MG63細胞的CTL細胞毒殺傷效應顯示各效靶濃度下CT+IL-2+HSP70組的殺傷活性高于CT+IL-2組,最高達56.33％±1.95％.結論 同時沉默Ezrin、過錶達HSP70既可以促進骨肉瘤細胞凋亡抑製其增殖,併利用HSP70誘導的CTL,增彊對靶腫瘤細胞的殺傷作用.
목적 탐토Ezrin기인침묵연합열휴극단백(heat shock protein,HSP)70유도적면역살상대골육류세포증식화조망적영향.방법 채용인성골육류MG63세포계작위연구대상,장인HSP70화Ezrin-shRNA적DNA편단분별극륭지함CMV화pU6쌍계동자적표체재체pGFP-V-RS중구건함HSP70화Ezrin-shRNA적진핵표체재체pGFP-V-RS-shRNA화pGFP-V-RS-shRNA-HSP70.중조질립분별전염입세포,사선출은정표체적세포극륭.형광현미경관찰세포형태급전염효과;형광정량RT-PCR급단백인적western blot검측은정전염세포주Eznn화HSP70기인급단백수평적변화;채용MTT、류식세포술검측세포증식、조망능력변화,Western blot검측조망급주기상관단백표체량변화;MTT법검측HSP70자격산생특이성세포독성T림파세포(CTL)대파종류세포적살상작용.결과 형광도상급기인화단백표체분석증실,수차성공구건동시침묵Ezrin화과표체HSP70적특이성재체.교단순침묵Ezrin,동시과표체HSP70회재일정정도상감약침묵Ezrin단백촉진세포조망화억제증식적효과,단여대조세포상비,M63세포조망솔잉명현상승,자11.01％±0.22％상승지24.28％±0.50％,증식속도칙자395.14％±2.24％감소지310.00％±2.83％.령외,Western blot검측발현Ezrin-shRNA가촉진조망기인Bax적표체,상반가강저항조망기인Bcl-2화세포주기단백Cyclin D1적표체.이Ezrin-shRNMHSP70조교Ezrin-shRNA조촉Bax표체화강저Bcl-2、세포주기단백Cyclin D1표체유소감약,단교음성대조조잉유명현효과.MG63세포적CTL세포독살상효응현시각효파농도하CT+IL-2+HSP70조적살상활성고우CT+IL-2조,최고체56.33％±1.95％.결론 동시침묵Ezrin、과표체HSP70기가이촉진골육류세포조망억제기증식,병이용HSP70유도적CTL,증강대파종류세포적살상작용.
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 LM 8 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.
