MicroRNA-93-5p与MicroRNA-20b-5p对VEGF基因转录调控作用与鹿茸细胞增殖关系的研究

发布时间：2018-04-09 05:21

本文选题：梅花鹿　切入点：微小RNA　出处：《吉林农业大学》2015年硕士论文

【摘要】：目前人们对鹿茸的研究涉及不同领域,其中主要分为以下几个方面:1、从鹿茸的基因及细胞活性角度研究其生长特性,揭示其起源。2、梅花鹿鹿茸的药理药化的研究。3、研究各类生长因子在鹿茸生长过程中所起到的重要作用。近年来,对鹿茸生长过程中相关生长因子的研究是热点,其中血管内皮生长因子(VEGF)在鹿茸的生长过程中起到重要的作用。VEGF是一种主要存在于血管细胞中的特异性有丝分裂原,其作用主要是通过与其受体(VEGFR)的结合来发挥介导微血管的增生及增强血管通透性等生理作用。其miRNA通过与3’非编码区(3’UTR)序列结合进行互补配对以期达到抑制靶基因表达的作用。本课题利用RNA基因组学技术,对miRNA介导的VEGF基因沉默在鹿茸软骨组织细胞增殖抑制作用的研究进行探讨,为研究VEGF对鹿茸快速生长再生的分子调控机制提供新思路。本实验将对梅花鹿鹿茸VEGF基因3’非编码区基因及其突变体序列进行克隆,运用课题组前期制备鹿茸顶端软骨和间充质组织的mi RNA芯片联合相关生物信息学软件进行初步筛选。运用荧光定量PCR技术及Western blotting进行进一步筛选确定能与VEGF3’UTR结合并发挥功能的miRNAs。构建双荧光素酶报告基因载体以检测相对荧光素酶活性,进而证实VEGF 3’UTR中miRNAs结合位点。荧光定量PCR检测经转染实验后,miRNAs在鹿茸细胞内的稳定性及相对表达水平。MTT法检测转染后鹿茸细胞的体外增殖状况。Western blotting检测经转染实验后,鹿茸细胞中VEGF蛋白的表达水平变化规律。ELISA法检测转染miRNAs鹿茸细胞中端粒酶的含量变化,同时探究VEGF与细胞因子bFGF及IGF的相关性。实验运用基因克隆技术成功获得了356bp梅花鹿VEGF 3’UTR野生型部分序列,梅花鹿与牛、人的VEGF基因3’UTR序列的核苷酸序列同源性经Blast分析比对分别为97.02%和88.76%,与牛的同源性较高。同时运用PCR定点突变技术以野生型为模板成功获得突变体序列全长336bp,成功将靶序列GCACTTT突变成TGTAGCG。miRNAs的初步筛选是应用生物信息学软件及miRNA基因芯片联合的方式进行,筛选出的显著差异表达的miRNAs共有8个。随后通过SYBR Green实时荧光定量PCR技术及Western blotting技术进行进一步筛选显示,miRNA-93-5p、mi RNA-20b-5p可与VEGF 3’UTR序列特异性结合,转染miRNA-93-5p、mi RNA-20b-5p后VEGF蛋白的表达水平抑制效果最为明显。经双酶切鉴定,实验成功构建双荧光素酶报告基因载体,双荧光素酶报告基因检测显示miRNA-93-5p、miRNA-20b-5p对VEGF基因有转录调控作用,VEGF是miRNA-93-5p、miRNA-20b-5p调控的靶基因;qPCR检测结果显示,转染miRNA-93-5p、miRNA-20b-5p mimics24h、48h、72h后,与对照组相比,高表达量的miRNA在转染组细胞中被检测到;MTT检测转染后梅花鹿茸细胞体外增殖,结果显示与对照组相比,转染miRNA-93-5p和miRNA-20b-5p组鹿茸细胞体外增殖受到抑制;运用ELISA法检测转染miRNA-93-5p、miRNA-20b-5p mimics后梅花鹿鹿茸细胞中端粒酶的含量,与对照组相比,转染组细胞端粒酶含量随着时间的延长而逐步降低;Western blotting检测转染后鹿茸细胞中VEGF蛋白的表达水平随时间的延长而降低,且转染后梅花鹿茸软骨细胞中VEGF的表达分别与bFGF、IGF的表达存在一定的相关性。
[Abstract]:The current research on velvet covering different areas, which is divided into the following aspects: 1. Study on the growth characteristics of gene and cell activity from the angle of pilose antler, reveals the origin of.2,.3 and pharmacological effects of the sika deer antler, the important role played by the research on all kinds of growth factors in the antler growth process. In recent years, the related growth factors in the process of antler growth is hot, among which vascular endothelial growth factor (VEGF) in the growth process of antler play a role in.VEGF is important for a specific exists mainly in vascular cells in mitogen, its function is mainly through its receptor (VEGFR). According to play mediated microvascular hyperplasia and enhanced vascular permeability. The physiological role of miRNA with 3 "non encoding region (3 UTR) combined with complementary sequences in order to inhibit target gene expression. This paper used RNA genomics technology on VEGF miRNA mediated gene silencing in inhibition of antler cartilage cell proliferation, and provide new ideas for the study of molecular VEGF on regeneration of antler rapid growth mechanism. In the experiment of sika deer antler VEGF gene 3 'non sequence gene and its mutant encoding cloning of MI RNA chip, combined with bioinformatics software using the previous system research group prepared antler tip cartilage and mesenchymal tissue were screened. Using fluorescence quantitative PCR and Western blotting were further screened to determine UTR and VEGF3' combination and function of the miRNAs. construction of dual luciferase reporter vector to detect the relative luciferase the activity then confirmed that VEGF miRNAs 3 'binding sites in UTR. Fluorescence quantitative PCR detection of transfected miRNAs cells after the experiment, in the antler Stability and relative expression in transfected cells was detected by.MTT.Western after the level of blotting in vitro proliferation of cells by transfection experiments to detect the antler, content changes of telomerase transfected miRNAs cells the expression level changes of.ELISA pilose antler cell method VEGF protein in the VEGF cells and explore the relationship between factor bFGF and IGF. The use of gene cloning technical success of the 356bp VEGF UTR '3 sika deer wild type sequence, sika deer and cattle by Blast analysis were 97.02% and 88.76% nucleotide sequences homologous to human VEGF gene 3' UTR sequence, and had high homology with cattle. At the same time using PCR mutagenesis with wild type mutant sequence successfully as template the full-length 336bp, successful target sequence of GCACTTT mutation into TGTAGCG.miRNAs screening is the application of bioinformatics software and miRNA microarray The way of expression, significant differences in the screening of miRNAs. A total of 8 followed by SYBR Green real-time fluorescence quantitative PCR technology and Western Blotting Technology for further screening showed that miRNA-93-5p, MI RNA-20b-5p and VEGF 3 UTR sequence specific binding of miRNA-93-5p mi RNA-20b-5p after transfection, the expression level of VEGF protein inhibitory effect of the obviously. Through double enzyme digestion experiment successfully constructed dual luciferase reporter gene vector, dual luciferase reporter gene assay showed that miRNA-93-5p and miRNA-20b-5p have effect on transcriptional regulation of VEGF gene, VEGF miRNA-93-5p, miRNA-20b-5p target gene regulation; qPCR assay showed that transfection of miRNA-93-5p miRNA-20b-5p, mimics24h, 48h, 72h, compared with the control group, high expression of miRNA was detected in the transfected cells; cell proliferation of antler MTT detection after transfection, and the results show Compared with the control group, miRNA-93-5p group and miRNA-20b-5p transfection inhibited cell proliferation of antler; using transfected miRNA-93-5p ELISA method, the content of telomerase of sika deer antler cells in miRNA-20b-5p mimics, compared with the control group, the content of telomerase group cells transfected with the time prolonged gradually decreased; the expression level of Western blotting VEGF after transfection was detected in antler cell protein decreased with the increase of time, and the expression of VEGF after transfection of antler chondrocytes in respectively with bFGF, there is a correlation between the expression of IGF.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S825;Q78

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