本文選題：非創(chuàng)傷性股骨頭壞死 + microRNA　； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：目的：觀察非創(chuàng)傷性股骨頭壞死患者與骨性關(guān)節(jié)炎患者骨髓基質(zhì)干細(xì)胞中主要miRNA的表達(dá)差異,尋找在非創(chuàng)傷性股骨頭壞死發(fā)病過(guò)程中起重要調(diào)節(jié)作用的miRNA。 方法：大量查閱文獻(xiàn)后找到參與調(diào)節(jié)骨髓基質(zhì)干細(xì)胞成骨分化,成脂分化,增殖相關(guān)過(guò)程的主要備選miRNA10個(gè),分別為：miR-20a, miR-17-5p, miR-27a-3p, miR-96-5p, miR-124-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p。臨床上收集了20例非創(chuàng)傷性股骨頭壞死患者原代骨髓基質(zhì)干細(xì)胞以及10例對(duì)照組患者原代骨髓基質(zhì)干細(xì)胞,使用實(shí)時(shí)熒光定量RT-PCR方法檢測(cè)上述10個(gè)miRNA的在實(shí)驗(yàn)組及對(duì)照組中的表達(dá)。 結(jié)果：miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p在實(shí)驗(yàn)組表達(dá)較對(duì)照組顯著降低,有統(tǒng)計(jì)學(xué)意義(p0.05),miR-96-5p, miR-124-3p在對(duì)照組和實(shí)驗(yàn)組間無(wú)明顯差異(p0.05)。 結(jié)論：miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p可能通過(guò)靶向于不同的mRNA調(diào)節(jié)骨髓基質(zhì)干細(xì)胞的分化與增殖,在非創(chuàng)傷性股骨頭壞死病理過(guò)程中起作用。 目的：miR-17-5p在非創(chuàng)傷性股骨頭壞死患者M(jìn)SC中表達(dá)水平顯著低于對(duì)照組,本部分實(shí)驗(yàn)?zāi)康脑谟谕ㄟ^(guò)生物信息學(xué)預(yù)測(cè)與熒光素酶報(bào)告基因檢測(cè)找到并確定miR-17-5p的靶基因,并檢測(cè)其在非創(chuàng)傷性股骨頭壞死患者M(jìn)SC標(biāo)本中的表達(dá)水平以及在HMSC-bm成骨分化過(guò)程中的表達(dá)水平變化規(guī)律。 方法：使用TargetScan6.2與Pictar網(wǎng)站進(jìn)行miR-17-5p靶基因預(yù)測(cè)。實(shí)時(shí)定量PCR檢測(cè)第一部分中20個(gè)非創(chuàng)傷性股骨頭壞死患者M(jìn)SC標(biāo)本中SMAD7表達(dá)水平,以及其與miR-17-5p表達(dá)水平是否具有相關(guān)性。實(shí)時(shí)定量PCR檢測(cè)BMP-2誘導(dǎo)的HMSC-bm成骨分化過(guò)程中第0,1,2,4,6天miR-17-5p及SMAD7表達(dá)水平變化趨勢(shì)(與不加入BMP-2的對(duì)照組進(jìn)行比較)。在HMSC-bm細(xì)胞中使用miR-17-5p類(lèi)似物與miR-17-5p抑制劑調(diào)整miR-17-5p表達(dá)水平,實(shí)時(shí)定量PCR檢測(cè)SMAD7表達(dá)水平,Western蛋白印跡試驗(yàn)檢測(cè)Smad7濃度。采用熒光素酶報(bào)告基因檢測(cè)方法,檢測(cè)miR-17-5p與SMDA7是否具有調(diào)控關(guān)系。 結(jié)果：SMAD7通過(guò)生物信息學(xué)預(yù)測(cè)可能是miR-17-5p的靶基因,結(jié)合SMAD7的生理作用我們認(rèn)為miR-17-5p可能通過(guò)靶向抑制SMAD7在非創(chuàng)傷性股骨頭壞死病理過(guò)程中起調(diào)控作用。Real-time PCR結(jié)果顯示在非創(chuàng)傷性股骨頭壞死患者M(jìn)SC標(biāo)本中miR-17-5p與SMAD7表達(dá)水平呈負(fù)相關(guān)(R2=0.5440,p=0.0002)。在BMP-2誘導(dǎo)的HMSC-bm成骨分化過(guò)程中miR-17-5p的表達(dá)水平自誘導(dǎo)后第一天起顯著高于對(duì)照組(p0.05),SMAD7的表達(dá)水平自誘導(dǎo)后第二天起顯著低于對(duì)照組(p0.05)。miR-17-5p類(lèi)似物組SMAD7表達(dá)水平與Smad7蛋白濃度顯著高于陰性對(duì)照組,miR-17-5p抑制劑組SMAD7表達(dá)水平與Smad7蛋白濃度顯著低于陰性對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。熒光素酶報(bào)告基因檢測(cè)示miR-17-5p通過(guò)靶向抑制SMAD7下調(diào)其表達(dá)。 結(jié)論：miR-17-5p通過(guò)不完全互補(bǔ)結(jié)合SMAD7的3'UTR端靶向抑制其表達(dá),在非創(chuàng)傷性股骨頭壞死發(fā)病過(guò)程及MSC成骨分化過(guò)程中發(fā)揮調(diào)控作用。 目的：研究]miR-17-5p提高HMSC-bm細(xì)胞系增殖與成骨分化的機(jī)制。 方法：在HMSC-bm細(xì)胞中轉(zhuǎn)染miR-17-5p類(lèi)似物與miR-17-5p抑制劑調(diào)整miR-17-5p表達(dá)水平并設(shè)立陰性對(duì)照,轉(zhuǎn)染4小時(shí)后加入100ng/ml BMP-2進(jìn)行成骨誘導(dǎo)。6天后實(shí)時(shí)定量PCR觀察RUNX2以及Ⅰ型膠原表達(dá)水平改變,通過(guò)ALP染色觀察HMSC-bm細(xì)胞內(nèi)堿性磷酸酶表達(dá)強(qiáng)度,通過(guò)CCK-8試劑盒檢測(cè)細(xì)胞增殖的改變。為了證明HMSC-bm細(xì)胞增殖及成骨分化增強(qiáng)是通過(guò)SMAD7表達(dá)水平下降而導(dǎo)致的,我們通過(guò)轉(zhuǎn)染pcDNA3.1-SMAD7人為提高SMAD7表達(dá)水平,觀察以上效應(yīng)是否減弱及逆轉(zhuǎn)。 結(jié)果：在BMP-2誘導(dǎo)的HMSC-bm成骨分化過(guò)程中,轉(zhuǎn)染miR-17-5p類(lèi)似物組RUNX2, COL1A1的表達(dá)水平顯著高于陰性對(duì)照組(p0.05),堿性磷酸酶表達(dá)顯著高于對(duì)照組,細(xì)胞增殖顯著高于對(duì)照組(p0.05)。轉(zhuǎn)染miR-17-5p抑制劑組RUNX2,Ⅰ型膠原的表達(dá)水平顯著低于陰性對(duì)照組(p0.05),堿性磷酸酶表達(dá)顯著低于對(duì)照組,細(xì)胞增殖顯著低于對(duì)照組(p0.05)。通過(guò)轉(zhuǎn)染pcDNA3.1-SMAD7人為提高SMAD7表達(dá)水平后可見(jiàn)以上效應(yīng)被部分削弱。 結(jié)論：miR-17-5p可提高HMSC-bm細(xì)胞的成骨分化及增殖,這種調(diào)控通過(guò)靶向抑制SMAD7表達(dá)起作用。
[Abstract]:Objective: To observe the difference in the expression of main miRNA in the bone marrow stromal cells of patients with non traumatic osteonecrosis of femoral head and osteoarthritis, and to find the important regulatory role of miRNA. in the pathogenesis of non traumatic femoral head necrosis.
Methods: after consulting a large number of literature, we found the main alternative miRNA10 to regulate the osteogenic differentiation, lipid differentiation and proliferation related processes of bone marrow stromal cells, which were miR-20a, miR-17-5p, miR-27a-3p, miR-96-5p, miR-124-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, and miR-199a-5p., 20 cases of non traumatic cases were collected. The original bone marrow stromal cells of the patients with femoral head necrosis and 10 cases of the primary bone marrow stromal cells in the control group were used to detect the expression of the 10 miRNA in the experimental group and the control group by real time fluorescence quantitative RT-PCR.
Results: the expression of miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p in the experimental group was significantly lower than that in the control group. There was a significant difference (P0.05), miR-96-5p, miR-124-3p between the control group and the experimental group (P0.05).
Conclusion: miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p may regulate the differentiation and proliferation of bone marrow stromal stem cells by targeting different mRNA, and play a role in the pathological process of non traumatic femoral head necrosis.
Objective: the expression level of miR-17-5p in the patients with non traumatic avascular necrosis of the femoral head was significantly lower than that of the control group. The purpose of this study was to find and determine the target gene of miR-17-5p through bioinformatics prediction and luciferase reporter gene detection, and to detect the expression level of the MSC in the non traumatic patients with femoral head necrosis and the expression of the target gene in the MSC specimens. The expression level of HMSC-bm during osteogenic differentiation.
Methods: TargetScan6.2 and Pictar sites were used to predict the target gene of miR-17-5p. Real-time quantitative PCR was used to detect the level of SMAD7 expression in the MSC specimens of 20 non traumatic patients with avascular necrosis of the femoral head, and whether it was related to the expression level of miR-17-5p. Real-time quantitative PCR was used to detect the number of BMP-2 induced HMSC-bm in the process of osteogenesis. 0,1,2,4,6 days miR-17-5p and SMAD7 expression level changes (compared with those that do not join BMP-2). MiR-17-5p analogues and miR-17-5p inhibitors are used in HMSC-bm cells to adjust miR-17-5p expression level, real-time quantitative PCR detection SMAD7 expression level, Western protein blot test for Smad7 concentration. Using luciferase reporter base The detection method is used to detect whether miR-17-5p has a regulatory relationship with SMDA7.
Results: SMAD7 may be the target gene of miR-17-5p through bioinformatics. Combining the physiological role of SMAD7, we believe that miR-17-5p may play a regulatory role in the pathological process of non traumatic femoral head necrosis by targeting SMAD7, and.Real-time PCR results show miR-17-5p and SMAD7 in MSC specimens of non traumatic patients with necrosis of the femoral head. The expression level was negatively correlated (R2=0.5440, p=0.0002). The expression level of miR-17-5p in BMP-2 induced HMSC-bm osteogenesis was significantly higher than that of the control group (P0.05) from the first day after induction. The expression level of SMAD7 was significantly lower than the control group (P0.05).MiR-17-5p analogue group SMAD7 expression level and Smad7 protein concentration from the control group (P0.05) in the second day after induction. Significantly higher than the negative control group, the level of SMAD7 expression and Smad7 protein concentration in the miR-17-5p inhibitor group were significantly lower than that of the negative control group, and the difference was statistically significant (P0.05). The luciferase reporter gene detection showed that the expression of miR-17-5p was down regulated by the target inhibition SMAD7.
Conclusion: miR-17-5p plays a regulatory role in the process of non traumatic avascular necrosis of the femoral head and the process of MSC osteogenesis in the process of non traumatic necrosis of the femoral head through incomplete complementarity and the inhibition of the expression of the 3'UTR end target of the SMAD7.
Objective: To study the mechanism of]miR-17-5p enhancing the proliferation and osteogenic differentiation of HMSC-bm cell line.
Methods: transfection of miR-17-5p analogue and miR-17-5p inhibitor in HMSC-bm cells to adjust miR-17-5p expression level and set up negative control. After 4 hours transfection, 100ng/ml BMP-2 was added to.6 days after.6 days to observe RUNX2 and the expression level of type I collagen, and ALP staining was used to observe the alkaline phosphatase in HMSC-bm cells. In order to prove that the proliferation of HMSC-bm cells and the enhancement of osteogenic differentiation are caused by the decrease of the expression level of SMAD7, the expression level of SMAD7 is enhanced by transfection of pcDNA3.1-SMAD7, and the effects of the above effects are observed to see if the above effects are weakened and reversed by the CCK-8 kit.
Results: in the process of BMP-2 induced HMSC-bm osteogenesis, the expression of COL1A1 was significantly higher than that of the negative control group (P0.05), and the expression of alkaline phosphatase was significantly higher than that of the control group (P0.05). The cell proliferation was significantly higher than that of the control group (P0.05). The expression of miR-17-5p inhibitor group was significantly lower than that of the control group (P0.05). The expression level of type I collagen was significantly lower. In the negative control group (P0.05), the expression of alkaline phosphatase was significantly lower than that of the control group, and the cell proliferation was significantly lower than that of the control group (P0.05). The above effects were partially weakened after transfection of pcDNA3.1-SMAD7 to improve the expression of SMAD7.
Conclusion: miR-17-5p can enhance the osteogenic differentiation and proliferation of HMSC-bm cells. This regulation can play a role in inhibiting SMAD7 expression by targeting.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R681.8

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