本文關(guān)鍵詞： 廣西巴馬小型豬 Ⅱ型糖尿病 miR-103/107 miR-122Caveolin-1　出處：《廣西大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：糖尿病是人類三大慢性疾病之一,以Ⅱ型糖尿病居多。Ⅱ型糖尿病主要是由于機(jī)體的胰島素抵抗作用進(jìn)而導(dǎo)致全身能量代謝紊亂的疾病,患者在患病后期會出現(xiàn)糖尿病腎病、心臟病等并發(fā)癥,嚴(yán)重威脅人類健康。近年來研究發(fā)現(xiàn),小分子miRNAs能夠參與Ⅱ型糖尿病的調(diào)控。本研究的目的是測定廣西巴馬小型豬miRNAs的組織表達(dá)譜;對廣西巴馬小型豬Ⅱ型糖尿病模型不同組織：niRNAs的表達(dá)差異進(jìn)行分析；探討miR-103/107在體外和體內(nèi)對Ⅱ型糖尿病的調(diào)控機(jī)制,為人類糖尿病的研究、治療提供新的靶點(diǎn)。參照miRBase公布的miR-103、107、122、143-5p基因序列,設(shè)計(jì)特異性的擴(kuò)增引物,克隆廣西巴馬小型豬miR-103、107、122、143-5p基因的成熟序列。利用實(shí)時(shí)熒光定量PCR測定巴馬小型豬miRNAs的組織表達(dá)譜。結(jié)果顯示巴馬小型豬miR-103、107、122、143-5p與miRBase公布的豬、人類、小鼠、綿羊、雞等物種序列一致,未出現(xiàn)堿基突變；miR-122在肝臟組織中特異性的表達(dá),且表達(dá)量顯著高于其他miRNAs(P0.05);miR-103、107、143-5p在肝臟、骨骼肌、脂肪組織中均有表達(dá),且miR-103在骨骼肌和脂肪組織中的表達(dá)量顯著高于miR-143-5p和miR-122(P0.05)。通過實(shí)時(shí)熒光定量PCR的方法測定T2DM模型發(fā)病組、未發(fā)病以及對照組的肝臟、骨骼肌、脂肪組織中miR-103、107、122的表達(dá)水平,結(jié)果顯示發(fā)病組肝臟組織中miR-122的表達(dá)量上調(diào),miR-103/107在發(fā)病組三個組織中的表達(dá)量均上調(diào)。因此篩選miR-103/107進(jìn)行后續(xù)研究。利用miRDanda、Targetscan網(wǎng)站以及文獻(xiàn)報(bào)道預(yù)測miR-103/107能夠與人和小鼠Caveolin-1基因的3'UTR區(qū)結(jié)合,存在三個結(jié)合位點(diǎn),與NCBI公布的豬Caveolin-1基因序列進(jìn)行比對,豬Caveolin-1基因3'UTR區(qū)存在一個miR-103/107結(jié)合位點(diǎn)。本試驗(yàn)克隆巴馬小型豬Caveolin-1基因包含3'UTR區(qū)域的1885 bp基因序列,與GenBank中公布的豬Caveolin-1基因3'UTR序列比對,結(jié)果顯示巴馬小型豬Caveolin-1基因3'UTR存在8處堿基突變,1處堿基缺失,1處堿基插入,同源性為99.6%,巴馬小型豬Caveolin-1基因3'UTR序列在1658-1715 bp存在一處miR-103/107的結(jié)合位點(diǎn)。本試驗(yàn)構(gòu)建pEGFP-C1-pre-103、pcDNA3.1(+)-EGFP-pre-103、pEGFP-C1-pre-107、 pcDNA3.1 (+)-EGFP-pre-107真核表達(dá)載體,轉(zhuǎn)染小鼠成肌細(xì)胞C2C12,實(shí)時(shí)熒光定量PCR檢測miR-103/107、Caveolin-1基因在細(xì)胞內(nèi)的表達(dá)情況。結(jié)果顯示C2C12細(xì)胞內(nèi)轉(zhuǎn)染pEGFP-C1-pre-103、 pcDNA3.1(+)-EGFP-pre-103真核表達(dá)載體后miR-103的表達(dá)量上調(diào),C2C12細(xì)胞內(nèi)轉(zhuǎn)染pEGFP-C 1-pre-107、pcDNA3.1(+)-EGFP-pre-107真核表達(dá)載體后miR-107的表達(dá)量上調(diào)；在C2C12細(xì)胞內(nèi)過表達(dá)miR-103/107后,Caveolin-1基因的表達(dá)量均下調(diào)。結(jié)果表明,在體外過表達(dá)miR-103/107能夠直接抑制靶基因Caveolin-1的表達(dá)水平。采用實(shí)時(shí)熒光定量PCR測定巴馬小型豬T2DM模型肝臟、骨骼肌、脂肪組織中Caveolin-l的表達(dá)水平。結(jié)果表明,巴馬小型豬發(fā)病組Caveolin-l在三個組織中的表達(dá)量均下調(diào)。對巴馬小型豬T2DM模型中發(fā)病組和未發(fā)病組Caveolin-l基因與miR-103/107的結(jié)合區(qū)域進(jìn)行PCR擴(kuò)增并測序,結(jié)果顯示在Caveolin-l基因與miR-103/107結(jié)合區(qū)域的1683 bp位點(diǎn)存在A/T突變,發(fā)病組有AA基因型豬3頭、AT基因型豬3頭,未發(fā)病組AT基因型豬1頭、TT基因型豬2頭。綜上所述,巴馬小型豬T2DM模型發(fā)病組肝臟組織miR-122表達(dá)量上調(diào)；miR-103/107在發(fā)病組肝臟、骨骼肌和脂肪組織中的表達(dá)量均上調(diào)。miR-103/107表達(dá)量的上調(diào),可以直接作用于靶基因Caveolin-l基因的3'UTR區(qū),抑制靶基因Caveolin-l基因mRNA水平的表達(dá)。Caveolin-l基因與miR-103/107結(jié)合區(qū)域內(nèi)1683 bp位點(diǎn)的A/T突變可能與Ⅱ型糖尿病的易感性有關(guān)。
[Abstract]:Diabetes is one of the three major chronic diseases in type II diabetes. Type II diabetes is mainly due to systemic energy metabolism disorder effect of insulin resistance in the body, can occur in patients with diabetic nephropathy in later stage of disease, heart disease and other complications, a serious threat to human health. In recent years, the study found that the regulation of small molecule miRNAs to be able to participate in type II diabetes. The purpose of this study is to express spectrum determination of Guangxi Bama miniature pig miRNAs organization; the Guangxi Bama miniature pig model of type II diabetes in different tissues: analysis of differentially expressed niRNAs; discuss the regulation mechanism of miR-103/107 on type II diabetes in vitro and in vivo, for the study of human diabetes, provide the target a new treatment. According to miR-103107122143-5p gene sequence published by miRBase, the specific primers design, cloning of Guangxi Bama minipig mi The mature sequence of R-103107122143-5p gene. The expression spectrum determination of Bama minipig miRNAs using real-time fluorescence quantitative PCR. Results showed that miR-103107122143-5p and miRBase released a pig, Bama miniature pig human, mouse, sheep, chicken and other species sequence, no base mutation; the expression of miR-122 in liver tissue specificity, and the expression of significantly higher than the other miRNAs (P0.05); miR-103107143-5p in liver, skeletal muscle, expressed in adipose tissue, and the expression of miR-103 in skeletal muscle and adipose tissues was significantly higher than that of miR-143-5p and miR-122 (P0.05). Determination of T2DM model onset group by real-time quantitative PCR, and the control group without the disease of liver, skeletal muscle, the expression level of miR-103107122 in adipose tissue, results show that the expression of miR-122 in liver disease group, miR-103/107 group of three groups in the incidence of The expression of the fabric were raised. Therefore, further research of screening miR-103/107. Use of miRDanda, Targetscan and miR-103/107 can predict the site reported in the literature and the human and mouse Caveolin-1 gene 3'UTR binding region, there are three binding sites, and porcine Caveolin-1 based NCBI released by sequence comparison, there is a region of Porcine Caveolin-1 gene 3'UTR miR-103/107 binding sites. This experiment cloned Bama minipig Caveolin-1 gene contains 1885 BP gene sequences of the 3'UTR region, and porcine Caveolin-1 gene sequences of 3'UTR published in GenBank, shows that Bama miniature pig Caveolin-1 gene 3'UTR had 8 nucleotide mutations, 1 deletion, 1 bases insertion, the homology of 99.6% binding sites in Bama the small pig Caveolin-1 gene sequence of 3'UTR miR-103/107 in the presence of a 1658-1715 BP. The test construct pEGFP-C1-pre-103 (+) -EGFP-pre, pcDNA3.1 -103, pEGFP-C1-pre-107, pcDNA3.1 (+) -EGFP-pre-107 eukaryotic expression vector was transfected into mouse myoblast C2C12, real-time fluorescence quantitative PCR to detect miR-103/107 expression of Caveolin-1 gene in the cells. The results showed that C2C12 cells transfected with pEGFP-C1-pre-103, pcDNA3.1 (+) -EGFP-pre-103 eukaryotic expression vector miR-103 was up-regulated after C2C12 cells. Transfection of pEGFP-C 1-pre-107, pcDNA3.1 (+) -EGFP-pre-107 eukaryotic expression up-regulated miR-107 vector; overexpression of miR-103/107 in C2C12 cells, the expression level of Caveolin-1 gene was down regulated in vitro. The results showed that overexpression of miR-103/107 can directly inhibit the expression level of target gene Caveolin-1. Determination of T2DM model of liver, Bama miniature pigs by real time fluorescence quantitative PCR in skeletal muscle, adipose tissue Caveolin-l expression. The results show that the small group Caveolin-l Bama pig disease The expression in the three tissues were cut. The combination of regional onset group Bama miniature pig T2DM model and incidence of group Caveolin-l and miR-103/107 gene were amplified by PCR and sequenced. The results showed that in the Caveolin-l gene and miR-103/107 combined with 1683 BP sites in the presence of A/T mutations, the incidence of group AA genotype of 3 pigs. AT genotypes of 3 pigs, incidence of AT genotype TT genotype of 1 pigs, 2 pigs. In summary, Bama miniature pig T2DM model onset group liver tissue miR-122 expression; miR-103/107 expression in the pathogenesis of liver, skeletal muscle and adipose tissue was significantly up-regulated the expression of.MiR-103/107 increases, can direct effect of 3'UTR on the target region of Caveolin-l gene,.Caveolin-l gene expression and inhibition of miR-103/107 target gene Caveolin-l gene mRNA level combined with area of 1683 BP loci of A/T mutation may be associated with type II diabetes The susceptibility of the disease is related.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1

【參考文獻(xiàn)】

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本文編號：1491264