贵州医科大学学报

2015, v.40;No.177(06) 581-587

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慢病毒载体介导的RNA干扰对HDAC2基因表达的影响
The Inhibitation Expression of HDAC2 Gene Intereferred by Lentivirus Mediated RNA

刘燕青;黄健;黄韻祝;娄方方;孔维莹;
LIU Yanqing;HUANG Jian;HUANG Yunzhu;LOU Fangfang;KONG Weiying;Department of Medical Laboratory,Guizhou Medical University;Department of Biochemistry,Affiliated Hospital of Guizhou Medical University;

摘要(Abstract):

目的:构建组蛋白去乙酰化酶2(HDAC2)基因的慢病毒表达载体,研究HDAC2基因在肝癌Hep G2细胞中的表达。方法:将前期构建并鉴定正确的3条重组表达质粒p LKO.1-HDAC2-shRNA 1组、2组、3组及1条阴性对照p LKO.1-HDAC2-shRNAneg和空载体对照p LKO.1,运用脂质体法分别与包装质粒ps PAX2和包膜质粒p MD2.G共同转染人胚肾细胞293T,制备具有感染能力慢病毒颗粒;收集、纯化含病毒颗粒的上清液并感染人肝癌Hep G2细胞,设未处理Hep G2细胞为空白对照,采用Real time PCR和Western blotting检测感染48 h和72 h后Hep G2细胞中HDAC2基因mRNA和蛋白表达水平,鉴定、分析重组质粒的干扰效果。结果:与空白对照组相比,3条干扰序列转染肝癌Hep G2细胞后均能明显抑制HDAC2 mRNA的表达(P<0.05),其中以p LKO.1-HDAC2-shRNA 1组的抑制率更明显,达到82.09%;3条干扰序列均能明显抑制HDAC2蛋白的表达(P<0.05),以p LKO.1-HDAC2-shRNA 1组和3组的干扰效果最明显,但两者相比较差异无统计学意义(P>0.05)。结论:构建的重组质粒p LKO.1-HDAC2-shRNA能从转录水平和蛋白水平有效抑制HDAC2基因在Hep G2细胞中表达。
Objective: To constuct the HDAC2 gene lentivirus vector and to verify the expression of HDAC2 gene silencing in hepatocellular carcinoma Hep G2 cells. Methods: Correctly identified three pairs of recombinant plasmid p LKO. 1-HDAC2-shRNA1,2,3,one pair of negative control p LKO. 1-HDAC2-shRNAneg and empty plasmid p LKO. 1 were co-transfected respectively with packed plasmids ps PAX2 and envelope plasmid p MD2. G into human embryonic kidney cells 293 T to produce infectious lentiviral particles by liposome method. The supernatant containing virus particles were collected,purified and infected with Hep G2 cells. Untreated Hep G2 cells were blank control group. HDAC2 gene mRNA and protein expression levels in Hep G2 cells were detected by Real-time PCR and Western blotting respectively 48 h and 72 h after infection. The interfering effect of recombinant plasmids in each group was indentified and analyzed. Results: Real-time PCR results showed that compared with control group,3 interference sequences transfected into Hep G2 liver cancer cells could significantly inhibit HDAC2 mRNA expression( P < 0. 05),of which p LKO. 1-HDAC2-shRNA1 showed more obvious inhibition rate,reaching 82. 09%. Western blotting test results showed that 3 interference sequences could significantly inhibit HDAC2 protein expression( P < 0. 05),of which p LKO. 1-HDAC2-shRNA1 and p LKO. 1-HDAC2-shRNA3 showed more obvious interference effect,and the difference between them was not statistically significant( P > 0. 05). Conclusion: The constructed recombinant plasmid p LKO. 1-HDAC2-shRNA can effectively restrain HDAC2 gene expression in Hep G2 cells in the transcription level and protein level,which provides a useful tool for follow up study on HDAC2 gene function.

关键词(KeyWords): 组蛋白去乙酰化酶2;RNA干扰;肝癌Hep G2细胞;慢病毒载体;p LKO.1载体
histone deacetylase2;RNA interference;hepatocellular carcinoma HepG2 cells;lentivirus vector;pLKO.1vector

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基金项目(Foundation): 贵州省联合基金[黔科合LH字(2014)7114]

作者(Author): 刘燕青;黄健;黄韻祝;娄方方;孔维莹;
LIU Yanqing;HUANG Jian;HUANG Yunzhu;LOU Fangfang;KONG Weiying;Department of Medical Laboratory,Guizhou Medical University;Department of Biochemistry,Affiliated Hospital of Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2015.06.008

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