本文選題：miR-590-3p　切入點：間充質(zhì)干細胞　出處：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：本課題通過基于基因表達譜芯片的生物信息學(xué)數(shù)據(jù)挖掘間充質(zhì)干細胞成骨向分化過程中表達上調(diào)的潛在研究位點,并以qRT-PCR驗證,選取microRNA-590-3p作研究,結(jié)合信號通路網(wǎng)絡(luò)預(yù)測其作用靶點。利用慢病毒轉(zhuǎn)染構(gòu)建穩(wěn)定miR-590-3p過表達細胞株,與空載體人間充質(zhì)干細胞系及l(fā)ipofectamine2000瞬轉(zhuǎn)miR-590-3p抑制細胞系相對比,證明miR-590-3p對間充質(zhì)干細胞增殖、細胞周期調(diào)節(jié)、成骨向分化具有促進作用。并通過雙熒光素酶實驗驗證miR-590-3p促進人間充質(zhì)干細胞成骨向分化機制是通過與APC mRNA3'UTR特異序列結(jié)合進而調(diào)節(jié)Wnt/β-catenin信號通路。這對牙槽骨再生及頜骨缺損重建的骨組織工程學(xué)有一定的指導(dǎo)作用。第一章間充質(zhì)干細胞分化過程中microRNA表達譜的生物信息學(xué)分析目的:通過生物信息學(xué)手段挖掘潛在研究靶點,并進行初步驗證方法:利用生物信息學(xué)分析篩選潛在miRNAs,以qRT-PCR驗證誘導(dǎo)間充質(zhì)干細胞成骨向分化對其表達的影響,從中篩選待研究因子,進一步結(jié)合GO分析以及信號通路網(wǎng)絡(luò)分析探索潛在靶點。結(jié)果:篩選得到miR18,miR22,miR-590-3p等miRNA與成骨向分化相關(guān)。miR-590-3p隨成骨向分化誘導(dǎo),在人間充質(zhì)干細胞中表達明顯上升。靶基因預(yù)測為APC,FZD 1,Wnt5a等Wnt通路相關(guān)蛋白。結(jié)論:miR-590-3p與間充質(zhì)干細胞成骨向分化有關(guān),其調(diào)控通路可能為Wnt信號通路。第二章 miR-590-3p過表達/抑制人間充質(zhì)干細胞株的建立及生物學(xué)效應(yīng)研究目的:構(gòu)建并探討miR-590-3p過表達/抑制間充質(zhì)干細胞系對間充質(zhì)干細胞增殖和細胞周期調(diào)節(jié)的影響。方法:構(gòu)建miR-590-3p過表達細胞株、表達抑制細胞系和對照細胞系,利用MTT實驗評估三組細胞增殖能力,并通過檢測CyclinD1以及pRb磷酸化水平評估三組細胞對細胞周期調(diào)節(jié)的影響。結(jié)果:miR-590-3p過表達細胞株MTT染色吸光度高,CyclinD1以及pRb磷酸化水平表達增強,miR-590-3p表達抑制細胞系相反。結(jié)論:miR-590-3p對間充質(zhì)干細胞增殖、細胞周期調(diào)節(jié)具有促進作用。第三章 MicroRNA-590-3p影響人間充質(zhì)干細胞成骨向分化的相關(guān)機制研究目的:評價miR-590-3p對間充質(zhì)干細胞成骨向分化的影響,并驗證其靶基因。方法:qRT-PCR比較成骨相關(guān)因子在不同細胞系間表達水平的高低。利用茜素紅染色對比不同細胞系礦化沉積活動的強弱。通過TOP/FOP實驗評價miR-590-3p表達水平與wnt/β-catenin信號通路活性之間關(guān)聯(lián)。利用雙熒光素酶實驗驗證miR-590-3p與APC的mRNA序列特異性結(jié)合。結(jié)果:miR-590-3p表達水平與成骨相關(guān)因子表達水平及礦化沉積呈正相關(guān)。TOP/FOP實驗提示wnt/β-catenin信號通路水平在miR-509-3p過表達細胞株出現(xiàn)上調(diào),雙熒光素酶實驗提示APC的3'UTR是miR-590-3p特異性綁定位點。結(jié)論:miRNA-590-3p對間充質(zhì)干細胞成骨向分化有促進作用,這一作用與Wnt/β-catenin信號通路相關(guān),而miR-590-3p對于APC的轉(zhuǎn)錄后調(diào)節(jié)是其中重要的機制之一。
[Abstract]:Based on bioinformatics data based on gene expression microarray, the potential sites for up-regulation of expression in osteogenic differentiation of mesenchymal stem cells (MSCs) were explored in this study. MicroRNA-590-3p was selected to study by qRT-PCR validation. The stable miR-590-3p overexpression cell line was constructed by lentivirus transfection, and compared with empty vector human mesenchymal stem cell line and lipofectamine2000 transient miR-590-3p cell line, the proliferation of mesenchymal stem cells was proved by miR-590-3p. Cell cycle regulation, The mechanism of miR-590-3p promoting osteogenic differentiation of human mesenchymal stem cells is to regulate the Wnt/ 尾 -catenin signaling pathway by binding to the specific sequence of APC mRNA3'UTR. Bone tissue engineering for reconstruction of maxillary defects. Chapter 1 bioinformatics analysis of microRNA expression profile during the differentiation of mesenchymal stem cells objective: to explore potential targets by bioinformatics. The primary verification methods were as follows: bioinformatics analysis was used to screen potential miRNAs. qRT-PCR was used to verify the effect of inducing osteogenic differentiation of mesenchymal stem cells on the expression of MIRNAs.The factors to be studied were screened. Results: miRNA such as miR18 miR22miR-590-3p was found to be related to osteogenic differentiation. MiR-590-3p was induced by osteogenesis. The target genes were predicted to be Wnt transduction related proteins such as APC-FZD1Wnt5a. Conclusion the expression of Wnt pathway is related to the osteogenic differentiation of mesenchymal stem cells. The regulatory pathway may be Wnt signaling pathway. Chapter 2 Establishment and Biological effects of miR-590-3p overexpression / inhibition of Human Mesenchymal Stem Cell Lines objective: to construct and study miR-590-3p overexpression / inhibition of Mesenchymal Stem Cell Lines against Mesenchymal Stem cells. Effects of stem cell proliferation and cell cycle regulation. Methods: miR-590-3p overexpression cell lines were constructed. Expression inhibition cell line and control cell line were used to evaluate the proliferation ability of three groups of cells by MTT assay. The effects of CyclinD1 and pRb phosphorylation on cell cycle regulation were evaluated by detecting the phosphorylation level of CyclinD1 and pRb. Results the cell line MTT staining and pRb phosphorylation enhanced the expression of miR-590-3p to inhibit cell cycle regulation. Conclusion the proliferation of mesenchymal stem cells was induced by 10% miR-590-3p. Effects of MicroRNA-590-3p on osteogenic differentiation of human mesenchymal stem cells objective: to evaluate the effect of miR-590-3p on osteogenic differentiation of human mesenchymal stem cells. Methods the expression level of osteoblast-associated factors in different cell lines was compared by using the proportion of qRT-PCR. The mineralized deposition activity of different cell lines was determined by alizarin red staining. The expression level of miR-590-3p was evaluated by TOP/FOP assay. The activity of wnt/ 尾 -catenin signaling pathway was correlated. The specific binding of miR-590-3p to APC mRNA sequence was verified by double luciferase experiment. Results the expression level of miR-590-3p / MIR-590-3p was positively correlated with the expression level of osteoblast-related factors and mineralized deposition. Topp / FOP experiment showed that wnt/ 尾 -catenin was positively related to the expression of APC. Signal pathway level was up-regulated in miR-509-3p overexpression cell lines. Double luciferase assay suggested that 3'UTR of APC is a specific binding site for miR-590-3p. Conclusion: miRNA-590-3p can promote the osteogenic differentiation of mesenchymal stem cells, which is related to the Wnt/ 尾 -catenin signaling pathway. The posttranscriptional regulation of APC by miR-590-3p is one of the important mechanisms.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R68

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