發(fā)布時(shí)間：2018-03-13 21:51

  本文選題：萎縮性骨不連　切入點(diǎn)：芯片　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2011年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 miRNA (micro ribonucleic acid,微小核糖核酸)是一種單鏈非編碼小分子RNA(ribonucleic acid,核糖核酸),在基因表達(dá)調(diào)控方面具有廣泛和普遍的作用,但目前對(duì)其在骨折愈合與不愈合中的調(diào)控作用尚知之甚少。本研究擬首先篩選萎縮性骨不連修復(fù)組織中異常表達(dá)的miRNA種類(lèi),并通過(guò)生物信息學(xué)方法預(yù)測(cè)表達(dá)上調(diào)miRNA的成骨功能相關(guān)靶基因,再?gòu)募?xì)胞水平驗(yàn)證萎縮性骨不連組織中表達(dá)上調(diào)miRNA對(duì)預(yù)測(cè)靶基因的調(diào)控作用,從而闡明miRNA在萎縮性骨不連發(fā)病過(guò)程中的調(diào)控作用及其分子生物學(xué)機(jī)制,為骨不連的基礎(chǔ)和臨床研究提供理論和實(shí)踐依據(jù)。 方法 (1)以臨床患者萎縮性骨不連修復(fù)組織(A組,3例)與正常骨折愈合后內(nèi)固定鋼板取出時(shí)鋼板周?chē)丘杞M織(B組,3例)為研究對(duì)象,采用Exiqon miRCURYTM LNA microRNA芯片(11.0版)對(duì)各組織中提取的RNA進(jìn)行miRNA篩選分析。在篩選出萎縮性骨不連組織中異常表達(dá)的miRNA后,采用熒光qRT-PCR(quantitative real-time polymerase chain reaction,實(shí)時(shí)定量聚合酶鏈反應(yīng))對(duì)芯片結(jié)果中A組表達(dá)上調(diào)的miRNA在兩種組織的表達(dá)水平進(jìn)行驗(yàn)證,并采用瀏覽計(jì)算機(jī)預(yù)測(cè)數(shù)據(jù)庫(kù)的生物信息學(xué)方法預(yù)測(cè)其可能的靶基因,探尋miRNA可能參與的骨折愈合和萎縮性骨不連相關(guān)病理生理過(guò)程。 (2)上調(diào)miRNA與預(yù)測(cè)靶基因關(guān)系的驗(yàn)證：取人骨髓全血,離心分離hBMSCs (human bone mesenchymal stem cells,人骨髓基質(zhì)干細(xì)胞),傳代培養(yǎng)。將人工合成的四種表達(dá)上調(diào)miRNA的雙鏈小RNA轉(zhuǎn)染入第四代hBMSCs,48小時(shí)后提取細(xì)胞總RNA,采用qRT-PCR、WB (Western Blotting,蛋白印跡)、雙熒光素酶報(bào)告基因檢測(cè)等多種方法,驗(yàn)證miRNA對(duì)預(yù)測(cè)靶基因的作用,確認(rèn)miRNA在萎縮性骨不連中的具體調(diào)控作用和分子生物學(xué)機(jī)制。 (3) miRNA和相應(yīng)靶基因在誘導(dǎo)hBMSCs成骨分化過(guò)程中表達(dá)水平及功能的相關(guān)研究：采用成骨誘導(dǎo)培養(yǎng)液誘導(dǎo)hBMSCs成骨分化的方法,觀察在誘導(dǎo)hBMSCs成骨分化過(guò)程中不同時(shí)間點(diǎn)(0,12h,1d,2d,4d,7d,14d)相應(yīng)miRNA、靶基因mRNA(messenger ribonucleic acid,信使核糖核酸)與蛋白表達(dá)水平的變化趨勢(shì),進(jìn)一步驗(yàn)證這些上調(diào)miRNA在誘導(dǎo)成骨分化中的調(diào)控作用,并探討成骨誘導(dǎo)培養(yǎng)液的誘導(dǎo)成分在此過(guò)程中促進(jìn)成骨分化的作用。 結(jié)果 (1)萎縮性骨不連修復(fù)組織相對(duì)鋼板周?chē)丘杞M織有9種miRNA發(fā)生1.5倍以上表達(dá)上調(diào)(hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p, hsa-miR-654-5p,以及5種hsa-miRPlus),9種miRNA發(fā)生1.5倍以上表達(dá)下調(diào)(hsa-let-7b*, hsa-miR-220b, hsa-miR-513a-3p, hsa-miR-551a,hsa-miR-576-5p,hsa-miR-1236,kshv-miR-K12-6-5p,以及2種hsa-miRPlus).qRT-PCR結(jié)果提示,數(shù)據(jù)庫(kù)中收錄并在A組表達(dá)上調(diào)的四種miRNA在組織水平的表達(dá)變化趨勢(shì)與芯片結(jié)果一致。生物信息學(xué)分析發(fā)現(xiàn),四種表達(dá)上調(diào)miRNA的預(yù)測(cè)靶基因包括多種成骨功能相關(guān)基因。 (2)分離培養(yǎng)的hBMSCs具有典型的干細(xì)胞形態(tài)特征,貼壁時(shí)間短,細(xì)胞融合快,體外擴(kuò)增容易,可以向成骨細(xì)胞誘導(dǎo),適合作為種子細(xì)胞用于后續(xù)的實(shí)驗(yàn)研究。在成功將上調(diào)miRNA的合成雙鏈小RNA轉(zhuǎn)染進(jìn)第四代hBMSCs48h后,提取細(xì)胞總RNA,qRT-PCR檢測(cè)表明,三種預(yù)測(cè)靶基因(hsa-miR-149*的預(yù)測(cè)靶基因ALPL、 hsa-miR-221的預(yù)測(cè)靶基因PDGFA和hsa-miR-654-5p的預(yù)測(cè)靶基因BMP2)的mRNA表達(dá)量受到明顯抑制,其余預(yù)測(cè)靶基因的mRNA無(wú)明顯下降；WB則表明上述三種預(yù)測(cè)靶基因的蛋白表達(dá)量均受到明顯抑制。雙熒光素酶報(bào)告基因檢測(cè)則證明三種預(yù)測(cè)靶基因的預(yù)測(cè)靶位點(diǎn)分別直接受到hsa-miR-149*、hsa-miR-221和hsa-miR-654-5p的靶向調(diào)控,其中ALPL的兩個(gè)預(yù)測(cè)靶位點(diǎn)只有第二個(gè)受到hsa-miR-149*的直接調(diào)控。 (3)誘導(dǎo)hBMSCs成骨分化過(guò)程中,得到確認(rèn)的成骨性靶基因ALPL.PDGFA和BMP2的mRNA和蛋白表達(dá)量在多數(shù)時(shí)間點(diǎn)有明顯增加,尤其以誘導(dǎo)分化后2～7天變化最為顯著(ALPL蛋白為1～7天),第14天時(shí)又有所下降。不同時(shí)間點(diǎn)的miRNA.靶基因mRNA和蛋白水平存在表達(dá)差異,其中hsa-miR-149*和hsa-miR-654-5p的變化趨勢(shì)與各自靶基因ALPL和BMP2的mRNA及蛋白表達(dá)水平總體上呈負(fù)相關(guān),而hsa-miR-221與PDGFA的變化趨勢(shì)無(wú)明顯負(fù)相關(guān)關(guān)系,兩者的關(guān)系有待進(jìn)一步研究。 結(jié)論 (1)萎縮性骨不連修復(fù)組織中存在miRNA的異常表達(dá)；四種在該組織中上調(diào)的miRNA(hsa-miR-149*、hsa-miR-221、hsa-miR-628-3p和hsa-miR-654-5p)有可能通過(guò)抑制多種成骨功能相關(guān)靶基因的表達(dá),在調(diào)控骨折向萎縮性骨不連發(fā)生發(fā)展中發(fā)揮重要作用。 (2)細(xì)胞水平驗(yàn)證表明：四種在組織水平上調(diào)的miRNA中,hsa-miR-149*與其預(yù)測(cè)的靶基因ALPL、hsa-miR-221與其預(yù)測(cè)的靶基因PDGFA、hsa-miR-654-5p與其預(yù)測(cè)的靶基因BMP2之間存在直接調(diào)控作用,異常上調(diào)的miRNA通過(guò)抑制成骨功能相關(guān)靶基因ALPL、PDGFA和BMP2的表達(dá)而限制其生物學(xué)功能,引起了萎縮性骨不連的發(fā)生發(fā)展。 (3)多種成骨功能相關(guān)靶基因的mRNA和蛋白表達(dá)水平在誘導(dǎo)hBMSCs成骨分化過(guò)程中大多數(shù)時(shí)間點(diǎn)發(fā)生了表達(dá)上調(diào),hsa-miR-149*和hsa-miR-654-5p與其相應(yīng)靶基因ALPL和BMP2的mRNA及蛋白表達(dá)變化趨勢(shì)呈負(fù)相關(guān),說(shuō)明這兩種miRNA在誘導(dǎo)成骨分化過(guò)程中與其靶基因mRNA和蛋白表達(dá)水平的調(diào)控密切相關(guān)。 簡(jiǎn)述之,本研究從萎縮性骨不連修復(fù)組織中找到具有關(guān)鍵調(diào)控作用的miRNA,發(fā)現(xiàn)有三種表達(dá)異常上調(diào)的miRNA通過(guò)抑制成骨功能相關(guān)靶基因引起了萎縮性骨不連的發(fā)生發(fā)展,其中兩種miRNA的表達(dá)水平與成骨功能相關(guān)靶基因的mRNA和蛋白表達(dá)水平密切相關(guān)。本研究可能將為萎縮性骨不連的診斷和治療提供新的理念。
[Abstract]:objective
MiRNA (micro ribonucleic acid, microRNA) is a single chain non encoding small molecule RNA (ribonucleic acid, ribonucleic acid), has extensive role in gene expression regulation, but its role in the regulation of fracture healing and nonunion is still poorly understood. Abnormal expression of this study firstly screening of atrophic nonunion tissue in miRNA type, and through the method of bioinformatics to predict bone functional target genes into upregulating the expression of miRNA, up-regulated the expression of miRNA in prediction of regulation of target genes from the cell level verification of atrophic bone nonunion tissue, so as to clarify the miRNA in atrophic bone nonunion control the role and molecular mechanism of the pathogenesis, provide theoretical and practical basis for nonunion and clinical research.
Method
(1) in patients with atrophic nonunion tissue (3 cases of group A) and remove the plate normal after fracture healing in the bone callus around the plate (group B, 3 cases) as the research object, using the Exiqon miRCURYTM LNA microRNA chip (11 Edition) miRNA screening analysis of each tissue extract the abnormal expression of RNA. In the screening of atrophic nonunion tissue in miRNA, qRT-PCR (quantitative real-time polymerase by fluorescence chain reaction and real-time quantitative polymerase chain reaction) to validate the expression level of A group was up-regulated in miRNA chip results in two kinds of tissues, and the biological information database browsing computer prediction the prediction of the possible target genes to explore the methodology, miRNA may be involved in the healing of fracture and nonunion related pathophysiological processes.
(2) to verify the relationship between the upregulation of miRNA and prediction of target genes: the human bone marrow blood, centrifugal separation (human bone mesenchymal hBMSCs stem cells, human bone marrow stromal stem cells), subculture. The artificial synthesis of four kinds of small double stranded RNA by upregulating the expression of miRNA in the fourth generation hBMSCs, 48 hours after the extraction of total cell RNA, by qRT-PCR WB (Western, Blotting, Western blot), dual luciferase reporter gene assay and other methods, to verify the miRNA target prediction function, confirm the miRNA in atrophic bonenonunion regulation and molecular mechanism.
(3) study of the expression level and function of miRNA and the corresponding target genes in hBMSCs induced osteogenic differentiation in the process of using osteogenic medium to induce hBMSCs differentiation into bone, observed at different time points in the process of osteogenic differentiation induced by hBMSCs (0,12h, 1D, 2D, 4D, 7d, 14d) corresponding miRNA, mRNA (messenger ribonucleic acid target gene, messenger ribonucleic acid) and protein expression level change trend, further validation of these induced upregulation of miRNA in the regulation of bone differentiation, and to explore the osteogenic medium to induce osteogenic differentiation of components in the process fluid.
Result
(1) atrophic nonunion relative plate callus tissues around 9 miRNA occurred more than 1.5 times up-regulated (hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p, hsa-miR-654-5p, and 5 hsa-miRPlus), 9 miRNA more than 1.5 times the expression (hsa-let-7b*, hsa-miR-220b, hsa-miR-513a-3p, hsa-miR-551a, hsa-miR-576-5p, hsa-miR-1236, kshv-miR-K12-6-5p 2 hsa-miRPlus.QRT-PCR), and the results suggest that the database and expression of four miRNA up-regulated in tissue expression level change trend consistent with microarray results in the A group. The bioinformatics analysis, four up-regulated miRNA expression predicted target genes including many osteogenic genes.
(2) hBMSCs isolated from the stem cells with typical morphological characteristics, adherent cell fusion time is short, fast, easy amplification, can be induced into osteoblasts, suitable as seed cells for subsequent experimental study. The success of the upregulation of miRNA synthesis of small double stranded RNA transfected into the fourth generation of hBMSCs48h. Extraction of total RNA cells, qRT-PCR assay showed that three predicted target genes (predicted target gene of BMP2 PDGFA and hsa-miR-654-5p hsa-miR-221 predicted target gene prediction of target gene ALPL, hsa-miR-149*) mRNA expression was inhibited, the predicted target gene of mRNA decreased significantly; WB showed that the expression volume were significantly inhibited three prediction of target gene protein. Dual luciferase reporter gene assay demonstrated that the predicted target sites three predicted target genes were directly affected by hsa-miR-149*, hsa-miR-221 and hsa-miR-654-5p target Only second of the two target loci of ALPL were directly regulated by hsa-miR-149*.
(3) hBMSCs induced osteogenic differentiation process, confirmed the expression of mRNA and protein of bone target gene ALPL.PDGFA and BMP2 in most of the time points increased, especially in the differentiation of 2~7 days after the most significant changes (ALPL protein for 1~7 days), fourteenth days have declined. At different time points the target genes of miRNA. mRNA and protein levels in the differential expression of mRNA and protein, the changing trend of hsa-miR-149* and hsa-miR-654-5p and their target genes ALPL and BMP2 expression level was negatively correlated, and the changing trend of hsa-miR-221 and PDGFA no significant negative correlation, the relationship between the two needs further study.
conclusion
(1) abnormal expression of miRNA exists in the atrophic nonunion tissue; four up-regulated in the tissues of miRNA (hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p and hsa-miR-654-5p) can inhibit a variety of expression of functional genes and in the regulation of fracture to atrophic bone nonunion plays an important role in the development.
(2) show that the four kinds of cells in tissue level up-regulated miRNA, ALPL hsa-miR-149* and its target gene prediction, target gene PDGFA hsa-miR-221 and forecast, there is a direct regulatory role between BMP2 hsa-miR-654-5p and its target gene prediction, abnormal upregulation of miRNA by inhibiting osteoblast function related target gene expression of PDGFA and ALPL. BMP2 the limit of its biological functions, causing atrophic nonunion.
(3) a variety of mRNA and protein in bone functional genes expression in hBMSCs induced osteogenic differentiation in most of the time point had expression of mRNA and protein expression of hsa-miR-149* and hsa-miR-654-5p and its target genes ALPL and BMP2 showed a negative correlation trend, indicating that the two miRNA in the induction of expression regulation in the process of the differentiation of bone and its target gene mRNA and protein are closely related.
Briefly, this research has found the key regulatory role of miRNA from atrophic nonunion tissue, found three abnormal expression upregulation of miRNA by inhibiting osteogenic functions of target genes causing atrophic nonunion development, closely related to the expression level of mRNA protein and the expression level of two miRNA with the osteogenic function of target genes. This study may provide new ideas for the atrophic nonunion of the diagnosis and treatment.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R683;R3411

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