TNF-α rs1800620、rs4645843对LX-2细胞株TNF-α含量的影响
|
摘要
目的:构建TNF-α rs1800620(SNP1)和TNF-α rs4645843(SNP2)稳转株的细胞模型,研究SNPs对LX-2细胞系内TNF-α含量的影响。方法:利用野生型TNF-α定点突变合成TNF-α SNP1/TNF-α SNP2突变型质粒,慢病毒包装后转染至LX-2细胞株构建稳转株,分别采用RT-qPCR和Western blot检测各重组细胞中TNF-α mRNA和蛋白水平。结果:基因测序表明突变型质粒构建成功,倒置显微镜拍照表明细胞有绿色荧光表达稳转株构建成功,RT-qPCR和Western blot检测结果表明与野生组比较,SNP1、SNP2均能显著降低TNF-α mRNA和蛋白水平(P <0. 05)。结论:稳定表达TNF-α /TNF-α SNP1/TNF-α SNP2 LX-2细胞模型构建成功,且SNP1、SNP2均能显著降低TNF-α mRNA和蛋白水平。
Objective: To construct the cell models of TNF-α rs1800620( SNP1) and TNF-α rs4645843( SNP2) stable transgenic lines and study the effects of SNPs on the content of TNF-α in LX-2 cell line. Methods: TNF-α SNP1/TNF-α SNP2 mutant plasmid was synthesized by wild-type TNF-α targeting and then transfected into LX-2 cell line after lentivirus packaging. RT-qPCR and Western blot were used to detect the mRNA and protein levels of TNF-α in each recombinant cell. Results: The gene sequencing showed that plasmids were successfully constructed. Photographs from an inverted microscope showed that the recombinant cells had stable expression of GFP. The results of RT-qPCR and Western blot showed that compared with the wild group,SNP1 and SNP2 could significantly reduce the mRNA( P < 0. 05) and protein( P < 0. 05) levels of TNF-α. Conclusion: The recombinant cells stably expressing TNF-α/TNF-α SNP1/TNF-α SNP2 LX-2 are constructed. SNP1 and SNP2 can significantly reduce the mRNA and protein levels of TNF-α in LX-2 cell line.
Objective: To construct the cell models of TNF-α rs1800620( SNP1) and TNF-α rs4645843( SNP2) stable transgenic lines and study the effects of SNPs on the content of TNF-α in LX-2 cell line. Methods: TNF-α SNP1/TNF-α SNP2 mutant plasmid was synthesized by wild-type TNF-α targeting and then transfected into LX-2 cell line after lentivirus packaging. RT-qPCR and Western blot were used to detect the mRNA and protein levels of TNF-α in each recombinant cell. Results: The gene sequencing showed that plasmids were successfully constructed. Photographs from an inverted microscope showed that the recombinant cells had stable expression of GFP. The results of RT-qPCR and Western blot showed that compared with the wild group,SNP1 and SNP2 could significantly reduce the mRNA( P < 0. 05) and protein( P < 0. 05) levels of TNF-α. Conclusion: The recombinant cells stably expressing TNF-α/TNF-α SNP1/TNF-α SNP2 LX-2 are constructed. SNP1 and SNP2 can significantly reduce the mRNA and protein levels of TNF-α in LX-2 cell line.
引文
1 Chasman D,Adams RM. Predicting the functional consequences of non-synonymous single nucleotide polymorphisms:structure-based assessment of amino acid variation 1[J]. Journal of Molecular Biology,2001,307(2):683-706
2 Uglialoro AM,Turbay D,Pesavento PA,et al. Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-αgene promoter[J]. Tissue Antigens,1998,52(4):359-367
3 Seki N,Yamaguchi K,Yamada A,et al. Polymorphism of the 5'-flanking region ofthe tumor necrosis factor(TNF)-alpha gene[J].Journal of Infectious Diseases,1999,180(3):880-883
4 Bayley JP,Ottenhoff T HM,Verweij CL. Is there a future for TNF promoter polymorphisms?[J]. Genes&Immunity,2004,5(5):315-329
5 Kroeger KM,Carville KS,Abraham LJ. The 308 tumor necrosis factor-αpromoter polymorphism effects transcription[J]. Molecular Immunology,1997,34(5):391-399
6 Wilson AG,Symons JA,Mcdowell TL,et al. Effects of a polymorphism in the human tumor necrosis factorαpromoter on transcriptional activation[J]. Proc Natl Acad Sci USA,1997,94(7):3195-3199
7 Bataller R,Brenner DA. Liver fibrosis[J]. Journal of Clinical Investigation,2005,115(2):209-218
8 Knittel T,Müller L,Saile B,et al. Effect of tumour necrosis factoralpha on proliferation,activation and protein synthesis of rat hepatic stellate cells[J]. Journal of Hepatology,1997,27(6):1067-1080
9 Qidwai T,Khan F. Tumour necrosis factor gene polymorphism and disease prevalence[J]. Scandinavian Journal of Immunology,2011,74(6):522-547
10 Wang JK,Feng ZW,Li YC,et al. Association of tumor necrosis factor-αgene promoter polymorphism at sites-308 and-238 with non-alcoholic fatty liver disease:a meta-analysis[J]. Journal of Gastroenterology&Hepatology,2012,27(4):670–676
11 Sghaier I,Zidi S,Mouelhi L,et al. The relationship between TNF alpha gene polymorphisms(238/308),TNF RII VNTR(p75)and outcomes of hepatitis B virus infection in tunisian population[J].Gene,2015,568(2):140-145
12 Shin SP,Kim NK,Kim JH,et al. Association between hepatocellular carcinoma and tumor necrosis factor alpha polymorphisms in South Korea[J]. World J Gastroenterol,2015,21(46):13064-13072
13 Jeng JE,Tsai JF,Chuang LY,et al. Tumor necrosis factor-α308. 2polymorphism is associated with advanced hepatic fibrosis and higher risk for hepatocellular carcinoma[J]. Neoplasia,2007,9(11):987-992
14 Dabhi B,Mistry KN. In silico analysis of single nucleotide polymorphism(SNP)in human TNF-αgene[J]. Meta Gene,2014,2:586-595
15 Bayley JP,Rooij HD,Elsen PJVD,et al. Functional analysis of linker-scan mutants spanning the 376,308,244,and 238 polymorphic sites of the tnf-αpromoter[J]. Cytokine,2001,14(6):316-323
16 Tuncbilek S,Aydin K,Hizel K. Vitamin D and tumor necrosis factor-alpha receptor gene polymorphisms in various hepatitis b clinical conditions in turkey[J]. Gastroenterology Research,2013,6(5):185-190
17 Somi MH,Najafi L,Noori BN,et al. Tumor necrosis factor-alpha gene promoter polymorphism in Iranian patients with chronic hepatitis B[J]. Indian Journal of Gastroenterology Official Journal of the Indian Society of Gastroenterology,2006,25(1):14-15
18 Mekinian A,Tamouza R,Pavy S,et al. Functional study of TNF-αpromoter polymorphisms:literature review and meta-analysis[J]. European Cytokine Network,2011,22(2):88-102
19 Squitti R,Siotto M,Bucossi S,et al. In silico investigation of the ATP7B,gene:insights from functional prediction of non-synonymous substitution to protein structure[J]. Free Radical Research Communications,2014,33(4):383-391
2 Uglialoro AM,Turbay D,Pesavento PA,et al. Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-αgene promoter[J]. Tissue Antigens,1998,52(4):359-367
3 Seki N,Yamaguchi K,Yamada A,et al. Polymorphism of the 5'-flanking region ofthe tumor necrosis factor(TNF)-alpha gene[J].Journal of Infectious Diseases,1999,180(3):880-883
4 Bayley JP,Ottenhoff T HM,Verweij CL. Is there a future for TNF promoter polymorphisms?[J]. Genes&Immunity,2004,5(5):315-329
5 Kroeger KM,Carville KS,Abraham LJ. The 308 tumor necrosis factor-αpromoter polymorphism effects transcription[J]. Molecular Immunology,1997,34(5):391-399
6 Wilson AG,Symons JA,Mcdowell TL,et al. Effects of a polymorphism in the human tumor necrosis factorαpromoter on transcriptional activation[J]. Proc Natl Acad Sci USA,1997,94(7):3195-3199
7 Bataller R,Brenner DA. Liver fibrosis[J]. Journal of Clinical Investigation,2005,115(2):209-218
8 Knittel T,Müller L,Saile B,et al. Effect of tumour necrosis factoralpha on proliferation,activation and protein synthesis of rat hepatic stellate cells[J]. Journal of Hepatology,1997,27(6):1067-1080
9 Qidwai T,Khan F. Tumour necrosis factor gene polymorphism and disease prevalence[J]. Scandinavian Journal of Immunology,2011,74(6):522-547
10 Wang JK,Feng ZW,Li YC,et al. Association of tumor necrosis factor-αgene promoter polymorphism at sites-308 and-238 with non-alcoholic fatty liver disease:a meta-analysis[J]. Journal of Gastroenterology&Hepatology,2012,27(4):670–676
11 Sghaier I,Zidi S,Mouelhi L,et al. The relationship between TNF alpha gene polymorphisms(238/308),TNF RII VNTR(p75)and outcomes of hepatitis B virus infection in tunisian population[J].Gene,2015,568(2):140-145
12 Shin SP,Kim NK,Kim JH,et al. Association between hepatocellular carcinoma and tumor necrosis factor alpha polymorphisms in South Korea[J]. World J Gastroenterol,2015,21(46):13064-13072
13 Jeng JE,Tsai JF,Chuang LY,et al. Tumor necrosis factor-α308. 2polymorphism is associated with advanced hepatic fibrosis and higher risk for hepatocellular carcinoma[J]. Neoplasia,2007,9(11):987-992
14 Dabhi B,Mistry KN. In silico analysis of single nucleotide polymorphism(SNP)in human TNF-αgene[J]. Meta Gene,2014,2:586-595
15 Bayley JP,Rooij HD,Elsen PJVD,et al. Functional analysis of linker-scan mutants spanning the 376,308,244,and 238 polymorphic sites of the tnf-αpromoter[J]. Cytokine,2001,14(6):316-323
16 Tuncbilek S,Aydin K,Hizel K. Vitamin D and tumor necrosis factor-alpha receptor gene polymorphisms in various hepatitis b clinical conditions in turkey[J]. Gastroenterology Research,2013,6(5):185-190
17 Somi MH,Najafi L,Noori BN,et al. Tumor necrosis factor-alpha gene promoter polymorphism in Iranian patients with chronic hepatitis B[J]. Indian Journal of Gastroenterology Official Journal of the Indian Society of Gastroenterology,2006,25(1):14-15
18 Mekinian A,Tamouza R,Pavy S,et al. Functional study of TNF-αpromoter polymorphisms:literature review and meta-analysis[J]. European Cytokine Network,2011,22(2):88-102
19 Squitti R,Siotto M,Bucossi S,et al. In silico investigation of the ATP7B,gene:insights from functional prediction of non-synonymous substitution to protein structure[J]. Free Radical Research Communications,2014,33(4):383-391