脂联素基因启动子变异的功能研究

发布时间：2018-11-29 14:00

【摘要】：目的：脂联素(Adiponectin)是脂肪细胞特异性分泌的一种蛋白。研究表明血清脂联素水平与代谢综合征和肿瘤相关,而脂联素基因启动子变异影响其功能的发挥。我们前期探讨了脂联素基因启动子区SNP多态性与云南汉族人群非小细胞肺癌(Non-Small Cell Lung Cancer, NSCLC)发生发展的相关性,发现SNP-12140G等位基因有可能是NSCLC的危险因素(OR=1.516; 95% CI:1.098-2.094)。为更深一步地阐明脂联素基因结构变异和mRNA的关系,在本研究中,我们将对脂联素基因启动子变异的功能进行初步探讨。方法：1.利用聚合酶链式反应(Polymerase Chain Reaction, PCR)扩增APM1基因启动子区；2.将APM1基因启动子区连接至萤火虫萤光素酶报告基因载体pGL4.11上,构建萤火虫萤光素酶报告基因重组质粒；3.利用多位点基因定点突变试剂盒对重组质粒进行基因突变,获得包含3个SNP (SNP-12140GA, SNP-11426AG, SNP-11377CG)不同组合的8个单倍体的萤火虫萤光素酶报告基因重组质粒。4.测序分析重组质粒是否突变成功；5.重组质粒与海肾荧光素酶报告基因pGL4.74共转染诱导分化第八天的小鼠前脂肪细胞3T3 L1；6.转染后48h检测萤火虫萤光素酶的活性以反映所连接的启动子变异的作用。结果：成功构建重组质粒,并完成基因定点突变；获得8中不同SNP组合的单倍体重组质粒；瞬时转染小鼠前脂肪细胞3T3L1,不同单倍型的重组质粒表达的荧光素酶报告基因活性具有差异；在8种重组质粒中,具有SNP-11377CG的C等位基因的单倍型重组质粒的萤火虫荧光素酶报告基因活性的表达量高于G等位基因的重组质粒；在SNP-11377CG等位基因为G的4种重组质粒中,SNP-12140GA的A等位基因的重组质粒所表达的萤火虫荧光素酶报告基因活性高于G等位基因的重组质粒；SNP-11426基因型与荧光素酶活性表达没有明显的关系。结论：脂联素基因启动子变异能够通过影响启动子的活性来影响脂联素基因的表达；脂联素基因启动子区SNP-11377CG和SNP-12140GA可改变脂联素启动子的功能；SNP-11426与脂联素启动子的功能无明显相关性。
[Abstract]:Objective: adiponectin (Adiponectin) is a protein secreted by adipocytes. Serum adiponectin levels were associated with metabolic syndrome and tumors, and adiponectin gene promoter mutations affected its function. We studied the association of adiponectin gene promoter SNP polymorphism with the occurrence and development of non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) in Yunnan Han population. We found that SNP-12140G allele may be a risk factor for NSCLC (OR=1.516;). 95% CI:1.098-2.094). In order to further elucidate the relationship between adiponectin gene structural variation and mRNA, we will explore the function of adiponectin gene promoter mutation in this study. Method 1: 1. The promoter region of APM1 gene was amplified by polymerase chain reaction (Polymerase Chain Reaction, PCR). The promoter region of APM1 gene was ligated to the vector pGL4.11 of firefly luciferase reporter gene, and the recombinant plasmid of luciferase reporter gene was constructed. The recombinant plasmids containing 8 haploid luciferase reporter genes containing three SNP (SNP-12140GA, SNP-11426AG, SNP-11377CG) combinations were obtained by using multi-locus site-directed mutagenesis kit. 4. Sequencing analysis showed that the recombinant plasmid was successfully mutated. The recombinant plasmid was co-transfected with the pGL4.74 gene of sea kidney luciferase reporter gene into mouse preadipocytes 3T3 L1 / 6 on the eighth day of differentiation. 48 hours after transfection, the luciferase activity of firefly was detected to reflect the mutation of the linked promoter. Results: the recombinant plasmids were successfully constructed, and the haploid recombinant plasmids with different SNP combinations were obtained. After transient transfection of mouse preadipocytes 3T3L1, the luciferase reporter gene activity of different haplotype recombinant plasmids was different. Among the eight recombinant plasmids, the activity of fluorescent luciferase reporter gene in the haplotype recombinant plasmid with C allele of SNP-11377CG was higher than that in the G allele. Among the four recombinant plasmids with SNP-11377CG allele G, the activity of luciferase reporter gene of SNP-12140GA A allele was higher than that of G allele. There was no significant correlation between SNP-11426 genotype and luciferase activity. Conclusion: adiponectin gene promoter mutation can affect the expression of adiponectin gene by affecting the activity of the promoter, and SNP-11377CG and SNP-12140GA of adiponectin gene promoter can change the function of adiponectin promoter. There was no significant correlation between SNP-11426 and adiponectin promoter function.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R3416

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