| 靶向增殖细胞核抗原shRNA的构建及其对HepG2细胞增殖与凋亡的影响 |
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| 引用本文： | 张小鹰,吴风云,何金花,廖晓莉,王威,蒋建伟. 靶向增殖细胞核抗原shRNA的构建及其对HepG2细胞增殖与凋亡的影响[J]. 生物化学与生物物理进展, 2010, 37(3): 278-287 |
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| 作者姓名： | 张小鹰 吴风云 何金花 廖晓莉 王威 蒋建伟 |
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| 作者单位： | 暨南大学医学院生化教研室;广东省佛山市南海区妇幼保健院检验科;浙江省温州市平阳县人民医院;广东省广州市番禺区中心医院检验科; |
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| 基金项目： | 广东省科技计划(2009B080701051); 广东省医学科研基金(A2007322)资助项目~~ |
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| 摘 要： | 为了探讨抑制增殖细胞核抗原(PCNA)基因表达对人肝癌HepG2细胞增殖及凋亡的影响,将前期筛选出的最佳siRNA序列转化为能表达其小发夹结构RNA(smallhairpinRNAs,shRNA)的DNA序列,并与pSilencer2.0-U6质粒定向连接,构建靶向PCNA基因的siRNA真核表达载体pShPCNA,经DNA测序证实与设计完全一致.随后采用WST法及克隆形成抑制观察细胞增殖抑制情况、划痕实验来观察细胞迁移能力,流式细胞术、Hoechest33258染色、细胞线粒体膜电位改变检测细胞凋亡.在转染HepG2细胞48h后,pShPCNA组细胞PCNAmRNA表达明显下调,并出现明显的增殖抑制作用,明显抑制细胞克隆的形成和细胞的迁移力,且呈剂量-效应关系.流式细胞术检测发现:pShPCNA组细胞明显阻滞于G0/G1期,并出现明显的亚二倍体凋亡峰,出现明显的早期凋亡细胞群.荧光显微镜检测表明,细胞线粒体膜电位降低,并且细胞出现核固缩、凋亡小体等凋亡形态学变化.上述结果表明,成功构建了靶向PCNA基因的siRNA真核表达载体pShPCNA,pShPCNA转染HepG2细胞48h后,能够显著抑制细胞的生长增...
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| 关 键 词： | RNA干扰 增殖细胞核抗原 真核细胞表达载体 HepG2细胞 肝癌 |
| 收稿时间： | 2009-09-27 |
| 修稿时间： | 2009-11-18 |
Construction of shRNA Targeting PCNA Gene and Its Effects on Proliferation and Apoptosis of HepG2 Cell Lines |
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| ZHANG Xiao-Ying,WU Feng-Yun,HE Jin-Hu,LIAO Xiao-Li,WANG Wei and JIANG Jian-Wei. Construction of shRNA Targeting PCNA Gene and Its Effects on Proliferation and Apoptosis of HepG2 Cell Lines[J]. Progress In Biochemistry and Biophysics, 2010, 37(3): 278-287 |
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| Authors: | ZHANG Xiao-Ying WU Feng-Yun HE Jin-Hu LIAO Xiao-Li WANG Wei JIANG Jian-Wei |
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| Affiliation: | ZHANG Xiao-Ying1,2),WU Feng-Yun1,3),HE Jin-Hua1,4),LIAO Xiao-Li1),WANG Wei1),JIANG Jian-Wei1)** (1) Department of Biochemistry,Medical College,Jinan University,Guangzhou 510630,China,2) Department of Laboratory,Nanhai Women , Children Health Hospital,Foshan 528200 China,3) Pingyang People's Hospital,Wenzhou 325400 China,4) Department of Laboratory,Panyu Center Hospital,Guangzhou 511400,China) |
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| Abstract: | In order to explore the function of PCNA gene on the proliferation and apoptosis of hepatocelluar carcinoma HepG2 cells, to provide the experimental evidence and a new tool to further explore the function of PCNA gene and the feasibility of its gene therapy, the eukaryotic expression vector targeting PCNA was constructed. According to the sequence screened in previous work, PCNA siRNA was converted into cDNA coding expression of shRNA (small hairpin RNA). The cDNA was synthesized and inserted into plasmid p... |
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| Keywords: | RNA interference proliferating cell nuclear antigen eukaryotic expression vector HepG2 cell line hepatocelluar carcinoma |
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