LC3B基因启动子在心肌梗死病人中的遗传和功能变异研究

发布时间：2018-04-21 02:32

本文选题：心肌梗死 + LC3B基因　；参考：《山东大学》2017年硕士论文

【摘要】：目的:研究心肌梗死病人和健康人群中LC3B基因启动子序列变异情况,针对变异序列构建重组质粒,通过检测双荧光素酶的活性,对LC3B基因启动子进行功能分析,探讨LC3B基因启动子序列变异与心肌梗死关联的生物学实质,为心肌梗死的遗传学分析提供理论依据,为临床上心肌梗死的预防、诊断、治疗提供新的思路。方法:采用大样本的病例对照研究方法对LC3B基因启动子进行基因序列分析和功能测定,收集就诊于济宁医学院附属医院经诊断为急性心肌梗死的病人383例,同期于济宁医学院附属医院体检的人群中随机选取390例健康人为对照组,性别、年龄在两组间无显著差异,分别统计两组人群的临床资料。分别从病人组和对照组人群外周血中提取基因组DNA,根据LC3B基因的基因组序列,设计合成LC3B基因启动子引物,采用普通PCR方法扩增两组人群中LC3B基因启动子序列,对DNA序列直接测序,分析两组人群中LC3B基因启动子序列变异情况。构建含LC3B基因启动子序列变异位点的pMD19-T载体,扩增后酶切和测序鉴定,亚克隆LC3B基因启动子序列至pGL3-Basic萤火虫荧光素酶报告基因载体,联同PRL-TK海肾荧光素酶报告基因载体瞬时转染HEK293细胞和H9C2细胞,收集细胞裂解液后,采用双荧光素酶报告基因检测试剂盒检测两种细胞中萤火虫荧光素酶和海肾荧光素酶的活性,初步探讨LC3B基因启动子序列变异对其转录活性的影响,及在H9C2细胞和HEK293细胞中的差异性转录活性。结果:1.该研究人群中发现25个DNA序列变异(DSVs),其中有12个单核苷酸多态性(SNPs)。在心肌梗死病人中,发现7个新颖DSVs,分别为g.4107TA,g.4325GC,g.4445delG,g.4597CG,g.4891CA,g.4903GT,g.4938CT和 3 个 SNPs,g.4137CT(rs575567442),g.4208CT(rs111626199)和 g.4853AG(rs77019223)。而这些DSVs在对照组中没有发现。在对照组人群中,发现5个新颖杂合 DSVs:g.4106CT,g.4224TC,g.4889CG,g.4904CG,g.5001AC,和 2 个 SNPs:g.4175GC(rs543058021),g.4494AC(rs577616524),这 7个DSVs只在对照组中出现。其他8个DSVs,包括7个SNPs:g.4068GA(rs 552929429),g.4280CT(rs1 1117269),g.4282CT(rs532744297),g.4433GA(rsl 42738317),g.4631CG(rs35227715),g.4774GA(rs189170289),g.4883CA(r x16944733),和1个新颖DSV:g.4488GC,在MI组和对照组中均有出现,且出现频率在两组中无区别(P0.05)。有趣的是,我们发现g.4883CA(rs16944733)和g.4889CG在发生频率上紧密连锁。2.在体外培养的HEK293细胞和H9C2细胞中,g.4107TA,g.4325GC,g.4597CG,g.4891CA,g.4903GT,g.4938CT和g.4137CT(rs575567442),g.4853AG(rs77019223),均能降低 LC3B 基因启动子的转录活性(P0.05),未发现其他DSVs和SNPs改变LC3B基因启动子的转录活性(P0.05)。结论:LC3B基因启动子序列变异可能改变其转录活性,进而影响LC3B基因的表达水平,以不同信号途径在心肌梗死的发生发展中发挥重要作用。
[Abstract]:Objective: to study the variation of LC3B gene promoter sequence in patients with myocardial infarction and healthy population. To construct recombinant plasmid and analyze the function of LC3B gene promoter by detecting the activity of double luciferase. To explore the biological essence of the association between LC3B gene promoter sequence variation and myocardial infarction, to provide a theoretical basis for genetic analysis of myocardial infarction, and to provide a new idea for clinical prevention, diagnosis and treatment of myocardial infarction. Methods: a large sample case-control study was conducted to analyze the gene sequence and function of LC3B gene promoter. 383 patients diagnosed as acute myocardial infarction at the affiliated Hospital of Jining Medical College were collected. In the same period, 390 healthy people were randomly selected as control group in the affiliated hospital of Jining Medical College. There was no significant difference in sex and age between the two groups. The clinical data of the two groups were statistically analyzed. Genomic DNAs were extracted from peripheral blood of patients and control groups respectively. According to the genomic sequence of LC3B gene, primers of LC3B gene promoter were designed and synthesized, and the promoter sequences of LC3B gene were amplified by ordinary PCR method. The DNA sequence was sequenced and the variation of LC3B gene promoter sequence was analyzed in the two groups. The pMD19-T vector containing mutation site of LC3B promoter sequence was constructed. After amplification and sequencing, the promoter sequence of LC3B gene was subcloned into the luciferase reporter gene vector of pGL3-Basic firefly. After transient transfection of HEK293 cells and H9C2 cells with PRL-TK sea kidney luciferase reporter gene vector, double luciferase reporter gene assay kit was used to detect the activity of luciferase and luciferase in both cells. To explore the effect of LC3B promoter sequence variation on its transcriptional activity and its differential transcriptional activity in H9C2 cells and HEK293 cells. The result is 1: 1. A total of 25 DNA mutations were found in the study population, of which 12 were single nucleotide polymorphisms (SNPs). Among the patients with myocardial infarction, 7 novel DSVswere found, namely, g.4107TAA, 4325GCng, 4445delGng, 44597CGP, 4891CAN, 4903GTg.4938CT, and 3 SNPsrs5767442g.4208CTs111626199, and g.4853AGRs7701923, respectively. These DSVs were not found in the control group. In the control group, five novel heterozygous DSVs: G. 4106CTN, g. 4224TCU, g. 4889CGN. 4904CGN. 5001AC, and two SNPs: G. 4175GCnrs543058021 and g.4494ACrs57761652424 were found. These 7 DSVs were only found in the control group. The other 8 DSVs, including 7 SNPs:g.4068GA(rs 552929429, rs1 1117269G. 4282CTS 53 2744297G. 4433GArsl 427383171CGS35227715g.4774GArs18902883CAr x 16944733GC, were found in MI group and control group, and the frequency was no difference between the two groups (P0.05). Interestingly, we found that g.4883CAA rs16944733) and g.4889CG are closely linked to frequency. 2. In both HEK293 cells and H9C2 cells cultured in vitro, the transcriptional activity of LC3B gene promoter P0.05 was decreased by G. 4107TAG. 4325GCCP4597CGN. 4891CGT. 4903GTG. 4938CT and g.4137CTrs575567442ng. 4853AGS770192323232323. No other DSVs and SNPs altered the transcriptional activity of LC3B gene promoter (P0.05N), and no other DSVs and SNPs could alter the transcriptional activity of LC3B gene promoter. Conclusion the mutation of the promoter sequence of the 10% LC3B gene may change its transcription activity, and then affect the expression level of LC3B gene, and play an important role in the occurrence and development of myocardial infarction through different signal pathways.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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