本文選題：梅花鹿　切入點：微小RNA　出處：《吉林農(nóng)業(yè)大學》2015年碩士論文

【摘要】：目前人們對鹿茸的研究涉及不同領域,其中主要分為以下幾個方面:1、從鹿茸的基因及細胞活性角度研究其生長特性,揭示其起源。2、梅花鹿鹿茸的藥理藥化的研究。3、研究各類生長因子在鹿茸生長過程中所起到的重要作用。近年來,對鹿茸生長過程中相關生長因子的研究是熱點,其中血管內(nèi)皮生長因子(VEGF)在鹿茸的生長過程中起到重要的作用。VEGF是一種主要存在于血管細胞中的特異性有絲分裂原,其作用主要是通過與其受體(VEGFR)的結(jié)合來發(fā)揮介導微血管的增生及增強血管通透性等生理作用。其miRNA通過與3’非編碼區(qū)(3’UTR)序列結(jié)合進行互補配對以期達到抑制靶基因表達的作用。本課題利用RNA基因組學技術,對miRNA介導的VEGF基因沉默在鹿茸軟骨組織細胞增殖抑制作用的研究進行探討,為研究VEGF對鹿茸快速生長再生的分子調(diào)控機制提供新思路。本實驗將對梅花鹿鹿茸VEGF基因3’非編碼區(qū)基因及其突變體序列進行克隆,運用課題組前期制備鹿茸頂端軟骨和間充質(zhì)組織的mi RNA芯片聯(lián)合相關生物信息學軟件進行初步篩選。運用熒光定量PCR技術及Western blotting進行進一步篩選確定能與VEGF3’UTR結(jié)合并發(fā)揮功能的miRNAs。構(gòu)建雙熒光素酶報告基因載體以檢測相對熒光素酶活性,進而證實VEGF 3’UTR中miRNAs結(jié)合位點。熒光定量PCR檢測經(jīng)轉(zhuǎn)染實驗后,miRNAs在鹿茸細胞內(nèi)的穩(wěn)定性及相對表達水平。MTT法檢測轉(zhuǎn)染后鹿茸細胞的體外增殖狀況。Western blotting檢測經(jīng)轉(zhuǎn)染實驗后,鹿茸細胞中VEGF蛋白的表達水平變化規(guī)律。ELISA法檢測轉(zhuǎn)染miRNAs鹿茸細胞中端粒酶的含量變化,同時探究VEGF與細胞因子bFGF及IGF的相關性。實驗運用基因克隆技術成功獲得了356bp梅花鹿VEGF 3’UTR野生型部分序列,梅花鹿與牛、人的VEGF基因3’UTR序列的核苷酸序列同源性經(jīng)Blast分析比對分別為97.02%和88.76%,與牛的同源性較高。同時運用PCR定點突變技術以野生型為模板成功獲得突變體序列全長336bp,成功將靶序列GCACTTT突變成TGTAGCG。miRNAs的初步篩選是應用生物信息學軟件及miRNA基因芯片聯(lián)合的方式進行,篩選出的顯著差異表達的miRNAs共有8個。隨后通過SYBR Green實時熒光定量PCR技術及Western blotting技術進行進一步篩選顯示,miRNA-93-5p、mi RNA-20b-5p可與VEGF 3’UTR序列特異性結(jié)合,轉(zhuǎn)染miRNA-93-5p、mi RNA-20b-5p后VEGF蛋白的表達水平抑制效果最為明顯。經(jīng)雙酶切鑒定,實驗成功構(gòu)建雙熒光素酶報告基因載體,雙熒光素酶報告基因檢測顯示miRNA-93-5p、miRNA-20b-5p對VEGF基因有轉(zhuǎn)錄調(diào)控作用,VEGF是miRNA-93-5p、miRNA-20b-5p調(diào)控的靶基因;qPCR檢測結(jié)果顯示,轉(zhuǎn)染miRNA-93-5p、miRNA-20b-5p mimics24h、48h、72h后,與對照組相比,高表達量的miRNA在轉(zhuǎn)染組細胞中被檢測到;MTT檢測轉(zhuǎn)染后梅花鹿茸細胞體外增殖,結(jié)果顯示與對照組相比,轉(zhuǎn)染miRNA-93-5p和miRNA-20b-5p組鹿茸細胞體外增殖受到抑制;運用ELISA法檢測轉(zhuǎn)染miRNA-93-5p、miRNA-20b-5p mimics后梅花鹿鹿茸細胞中端粒酶的含量,與對照組相比,轉(zhuǎn)染組細胞端粒酶含量隨著時間的延長而逐步降低;Western blotting檢測轉(zhuǎn)染后鹿茸細胞中VEGF蛋白的表達水平隨時間的延長而降低,且轉(zhuǎn)染后梅花鹿茸軟骨細胞中VEGF的表達分別與bFGF、IGF的表達存在一定的相關性。
[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.

【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S825;Q78

【參考文獻】

相關期刊論文 前1條

1 耿濤;楊福合;邢秀梅;褚文輝;孫紅梅;李春義;;Notch信號通路在體外培養(yǎng)的鹿茸干細胞中的表達[J];特產(chǎn)研究;2010年02期

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本文編號：1725034