shRNA稳定转染抑制氯离子通道蛋白1基因的表达对小鼠肝癌细胞的影响  

Study on the influence of inhibiting the expression of CLIC1 gene with shRNA on mouse hepatocellular carcinoma cell

作　　者：李荣宽[1] 唐建武[2] 张军[2] 张宇虹[3] 王绍清[2] 王波[2] 王梅[2] 李妙玲[2] 陈文静[2] 金艳玲[2] 王静文[2] 魏元怡[2] 

机构地区：[1]大连医科大学附属第二医院消化内科,辽宁省116023 [2]大连医科大学病理学教研室 [3]大连医科大学附属第二医院超声科,辽宁省116023

出　　处：《中华临床医师杂志（电子版）》2010年第6期721-727,共7页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(30772468)

摘　　要：目的将shRNA稳定转染至小鼠肝癌细胞株Hca-F中,使得氯离子通道蛋白1(CLIC1)基因表达抑制,观察肿瘤细胞的增殖、细胞周期、凋亡情况以及迁移能力的变化。方法设计并合成理论上最佳的shRNA序列,进而将其插入pGPU6/GFP/Neo质粒中,经测序和双酶切验证载体构建成功后以梭华-Sofast将DNA质粒稳定转染至小鼠肝癌细胞株Hca-F中,经Real-timeRT-PCR验证CLIC1mRNA表达下调后,应用CCK-8试剂盒和流式细胞仪检测干扰前后的Hca-F细胞,对比观察细胞的增殖、细胞周期和凋亡情况的变化,应用Transwell小室检验其迁移能力的变化。结果 pGPU6/GFP/Neo-shRNA对CLIC1mRNA抑制效果明显,抑制率达57%。经该表达质粒的干扰后,Hca-F细胞的增殖能力明显增强,停留于G2/M期的细胞明显增多,凋亡细胞显著减少,Transwell小室中穿膜细胞数量明显减少。结论 CLIC1基因具有抑制小鼠肝癌细胞的增殖和促进其凋亡的作用,并且参与细胞周期中G2/M期的调节,同时能够促进肿瘤细胞的迁移。Objective To study the change of proliferation,cell cycle,apoptosis and migrasion of Hca-F cell after its expression of CLIC1 gene was effectively inhibited by shRNA. Methods The theoretic most effective shRNA sequence was designed and constructed,and the corresponding sequences were inserted into pGPU6 /GFP/Neo plasmids to construct expression vector,after being identified via double enzyme-cutting method and DNA sequencing,the expression vector was stably transfected into Hca-F cells by Sofast reagent,and then detected by real-time reverse-transcription polymerase chain reaction ( real-time RT-PCR) to verify its efficiency. Using CCK-8 kit and flow cytometry to study the change of proliferation,cell cycle and apoptosis of Hca-F cells after the expression of CLIC1 gene was inhabited,at the same time,cell migration was assessed in vitro by transwell chamber. Results The expression plasmid of pGPU6 /GFP/Neo-shRNA-611 effectively inhabited the expression of CLIC1 mRNA,the inhabit ratio was 57% . After the CLIC1 gene was inhabited,the energy of proliferation of Hca-F cells was increased,the number of cells in G2 /M period increased and the mumber of apoptosis cells decreased remarkably. Conclusions CLIC1 gene plays an important role in supressing the proliferation,promoting apoptosis and regulating cell cycle in Hca-F cells,meanwhile,it can promote carcinoma cells' migration.

关 键 词：癌 肝细胞 细胞增殖 细胞凋亡 细胞运动 RNA 干扰 小鼠 

分 类 号：R735.7[医药卫生—肿瘤]