沉默GPC-3基因质粒构建及对HepG2细胞增殖的抑制作用  

Construction of silencing glypican-3 gene plasmids and inhibition of human HepG2 cell proliferation

作　　者：蔚丹丹[1] 姚敏[2] 陈洁[1] 严晓娣[1] 邱历伟[1] 姚登福[1] 

机构地区：[1]南通大学临床医学研究中心,南通226001 [2]南通大学医学院,南通226001

出　　处：《南通大学学报（医学版）》2012年第5期346-349,共4页Journal of Nantong University(Medical sciences)

基　　金：国家国际科技合作专项(2011ZR0003);南通市社会发展项目(HS2011012);南通大学研究生创新计划(YKC12015);江苏省卫生科技项目(H2009025)

摘　　要：目的:构建磷脂酰肌醇蛋白多糖(glypican,GPC)-3 shRNA质粒,观察GPC-3活化干扰对肝癌(HepG2)细胞的抑制作用。方法:设计与合成多条针对GPC-3序列的shRNA,插入pGPU6/GFP/Neo载体,构建、转染HepG2细胞、筛选高效质粒,观察沉默GPC-3表达对肝癌细胞增殖的抑制作用与机制。结果:经BamHⅠ或PstⅠ酶切鉴定,证实插入pGPU6/GFP/Neo载体GPC-3 shRNA序列正确,成功构建GPC-3 shRNA干扰质粒。以脂质体介导,将GPC-3shRNA1～4分别转染HepG2细胞,shRNA1沉默GPC-3 mRNA最高效率达89.3%,可有效抑制HepG2细胞增殖,在72 h达71.1%;shRNA1干扰使HepG2细胞周期阻滞在G1期,促进癌细胞发生凋亡(65.6%)。结论:shRNA可有效干预肝癌细胞GPC-3基因转录,促进凋亡并抑制增殖,GPC-3可望成为肝癌基因治疗的靶目标。Objective： To investigate the silencing glypican-3（GPC-3） transcription on the effect of human HepG2 cell proliferation by constructing GPC-3 short hairpin RNA（shRNA）.Methods： Few pairs of GPC-3 shRNA were designed,synthesized,and constructed according to GPC-3 sequence,inserted pGPU6/GFP/Neo vector,transformed into E.coli to screening and identify high efficiency plasmid.The effective sequence transfected into HepG2 cells for observing GPC-3 expression,respectively.Results： pGPU6/GFP/NeoGPC-3-shRNA1-4 plasmids were successfully constructed,and confirmed by enzymatic digestion with BamH Ⅰ or Pst Ⅰ.After HepG2 cells transfected with liposome mediated-shRNAs,the rate of silencing GPC-3 mRNA was up to 89.3 % by shRNA1.The proliferation rate of the transfected HepG2 cells was 71.1 % at 72 h in the GPC-3 shRNA1 group.The cell cycles after the cells transfected with shRNA were arrested in the G1 phase,and the apoptosis rate of HepG2 cells was increased to 65.6%.Conclusion： Specific GPC-3 shRNA might intervene effectively the gene transcription,inhibit HCC cell proliferation,and promote apoptosis.GPC-3 should be a potential target gene for HCC gene therapy.

关 键 词：肝细胞性肝癌 磷脂酰肌醇蛋白多糖-3 短发夹型RNA 转染 基因沉默 

分 类 号：R735.7[医药卫生—肿瘤]