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)干細胞中主要miRNA的表達差異,尋找在非創(chuàng)傷性股骨頭壞死發(fā)病過程中起重要調(diào)節(jié)作用的miRNA。 方法：大量查閱文獻后找到參與調(diào)節(jié)骨髓基質(zhì)干細胞成骨分化,成脂分化,增殖相關(guān)過程的主要備選miRNA10個,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)干細胞以及10例對照組患者原代骨髓基質(zhì)干細胞,使用實時熒光定量RT-PCR方法檢測上述10個miRNA的在實驗組及對照組中的表達。 結(jié)果：miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p在實驗組表達較對照組顯著降低,有統(tǒng)計學(xué)意義(p0.05),miR-96-5p, miR-124-3p在對照組和實驗組間無明顯差異(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可能通過靶向于不同的mRNA調(diào)節(jié)骨髓基質(zhì)干細胞的分化與增殖,在非創(chuàng)傷性股骨頭壞死病理過程中起作用。 目的：miR-17-5p在非創(chuàng)傷性股骨頭壞死患者MSC中表達水平顯著低于對照組,本部分實驗?zāi)康脑谟谕ㄟ^生物信息學(xué)預(yù)測與熒光素酶報告基因檢測找到并確定miR-17-5p的靶基因,并檢測其在非創(chuàng)傷性股骨頭壞死患者MSC標(biāo)本中的表達水平以及在HMSC-bm成骨分化過程中的表達水平變化規(guī)律。 方法：使用TargetScan6.2與Pictar網(wǎng)站進行miR-17-5p靶基因預(yù)測。實時定量PCR檢測第一部分中20個非創(chuàng)傷性股骨頭壞死患者MSC標(biāo)本中SMAD7表達水平,以及其與miR-17-5p表達水平是否具有相關(guān)性。實時定量PCR檢測BMP-2誘導(dǎo)的HMSC-bm成骨分化過程中第0,1,2,4,6天miR-17-5p及SMAD7表達水平變化趨勢(與不加入BMP-2的對照組進行比較)。在HMSC-bm細胞中使用miR-17-5p類似物與miR-17-5p抑制劑調(diào)整miR-17-5p表達水平,實時定量PCR檢測SMAD7表達水平,Western蛋白印跡試驗檢測Smad7濃度。采用熒光素酶報告基因檢測方法,檢測miR-17-5p與SMDA7是否具有調(diào)控關(guān)系。 結(jié)果：SMAD7通過生物信息學(xué)預(yù)測可能是miR-17-5p的靶基因,結(jié)合SMAD7的生理作用我們認為miR-17-5p可能通過靶向抑制SMAD7在非創(chuàng)傷性股骨頭壞死病理過程中起調(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MSC標(biāo)本中miR-17-5p與SMAD7表達水平呈負相關(guān)(R2=0.5440,p=0.0002)。在BMP-2誘導(dǎo)的HMSC-bm成骨分化過程中miR-17-5p的表達水平自誘導(dǎo)后第一天起顯著高于對照組(p0.05),SMAD7的表達水平自誘導(dǎo)后第二天起顯著低于對照組(p0.05)。miR-17-5p類似物組SMAD7表達水平與Smad7蛋白濃度顯著高于陰性對照組,miR-17-5p抑制劑組SMAD7表達水平與Smad7蛋白濃度顯著低于陰性對照組,差異均有統(tǒng)計學(xué)意義(p0.05)。熒光素酶報告基因檢測示miR-17-5p通過靶向抑制SMAD7下調(diào)其表達。 結(jié)論：miR-17-5p通過不完全互補結(jié)合SMAD7的3'UTR端靶向抑制其表達,在非創(chuàng)傷性股骨頭壞死發(fā)病過程及MSC成骨分化過程中發(fā)揮調(diào)控作用。 目的：研究]miR-17-5p提高HMSC-bm細胞系增殖與成骨分化的機制。 方法：在HMSC-bm細胞中轉(zhuǎn)染miR-17-5p類似物與miR-17-5p抑制劑調(diào)整miR-17-5p表達水平并設(shè)立陰性對照,轉(zhuǎn)染4小時后加入100ng/ml BMP-2進行成骨誘導(dǎo)。6天后實時定量PCR觀察RUNX2以及Ⅰ型膠原表達水平改變,通過ALP染色觀察HMSC-bm細胞內(nèi)堿性磷酸酶表達強度,通過CCK-8試劑盒檢測細胞增殖的改變。為了證明HMSC-bm細胞增殖及成骨分化增強是通過SMAD7表達水平下降而導(dǎo)致的,我們通過轉(zhuǎn)染pcDNA3.1-SMAD7人為提高SMAD7表達水平,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成骨分化過程中,轉(zhuǎn)染miR-17-5p類似物組RUNX2, COL1A1的表達水平顯著高于陰性對照組(p0.05),堿性磷酸酶表達顯著高于對照組,細胞增殖顯著高于對照組(p0.05)。轉(zhuǎn)染miR-17-5p抑制劑組RUNX2,Ⅰ型膠原的表達水平顯著低于陰性對照組(p0.05),堿性磷酸酶表達顯著低于對照組,細胞增殖顯著低于對照組(p0.05)。通過轉(zhuǎn)染pcDNA3.1-SMAD7人為提高SMAD7表達水平后可見以上效應(yīng)被部分削弱。 結(jié)論：miR-17-5p可提高HMSC-bm細胞的成骨分化及增殖,這種調(diào)控通過靶向抑制SMAD7表達起作用。
[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)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R681.8

【相似文獻】

相關(guān)期刊論文 前10條

1 王運濤;骨髓間充質(zhì)干細胞成骨分化調(diào)控的研究進展[J];國外醫(yī)學(xué)(生物醫(yī)學(xué)工程分冊);2003年01期

2 井燕;李良;李毅;陳孟詩;吳文超;陳槐卿;劉小菁;;力學(xué)應(yīng)變對大鼠骨髓間充質(zhì)干細胞增殖和成骨分化能力的影響[J];生物醫(yī)學(xué)工程學(xué)雜志;2006年03期

3 崔向榮;蘇偉;黃釗;覃萬安;;電磁場促進骨髓間充質(zhì)干細胞成骨分化的研究進展[J];中國醫(yī)學(xué)物理學(xué)雜志;2011年04期

4 彭飛;鄭亞東;徐西強;虞冀哲;吳華;;620nm低能量紅光對骨髓間充質(zhì)干細胞成骨分化的影響[J];激光生物學(xué)報;2011年03期

5 趙領(lǐng)洲;劉麗;吳織芬;;微米坑/納米管氧化鈦形貌對骨髓間充質(zhì)干細胞成骨分化的作用[J];醫(yī)學(xué)爭鳴;2012年04期

6 楊姣;夏雷;湯郁;陸化;費小明;;腫瘤壞死因子預(yù)處理促進骨髓間充質(zhì)干細胞成骨分化潛能[J];南京醫(yī)科大學(xué)學(xué)報(自然科學(xué)版);2014年03期

7 王晶;張洹;;-80℃一步法凍存對成人骨髓間充質(zhì)干細胞成骨分化能力的影響[J];廣東醫(yī)學(xué);2007年03期

8 陳翠平;羅新;;17β-雌二醇對大鼠骨髓間充質(zhì)干細胞成骨分化的作用[J];中國骨質(zhì)疏松雜志;2007年05期

9 ;骨髓間充質(zhì)干細胞的成骨分化[J];中國組織工程研究與臨床康復(fù);2010年10期

10 吳玉瓊;房兵;江凌勇;;間歇牽拉應(yīng)變對大鼠骨髓間充質(zhì)干細胞增殖與成骨分化的影響[J];醫(yī)用生物力學(xué);2011年06期

相關(guān)會議論文 前10條

1 鄭亮;鄭雅元;崔燎;劉鈺瑜;;四羥基二苯乙烯-2-O-β-D-葡萄糖苷對大鼠骨髓間充質(zhì)干細胞成骨分化的作用及機制研究[A];中華醫(yī)學(xué)會第七次全國骨質(zhì)疏松和骨礦鹽疾病學(xué)術(shù)會議論文匯編[C];2013年

2 吳江;陳槐卿;K-LP.SUNG;;鈦顆粒負荷影響骨髓間充質(zhì)干細胞成骨分化能力的機理[A];全國首屆青年復(fù)合材料學(xué)術(shù)交流會論文集[C];2007年

3 吳江;陳槐卿;李良;尹光福;K-L P．SUNG;;鈦顆粒負荷影響骨髓間充質(zhì)干細胞成骨分化能力的機理[A];中國復(fù)合材料學(xué)術(shù)研討會論文集[C];2005年

4 陳海嘯;洪盾;萬曉晨;李繼承;;腸毒素C聯(lián)合維生素C對骨髓間充質(zhì)干細胞成骨分化的影響研究[A];2008年浙江省骨科學(xué)學(xué)術(shù)年會論文匯編[C];2008年

5 袁風(fēng)紅;鄒耀紅;高愷言;俞可佳;;地塞米松對體外人骨髓基質(zhì)細胞增殖及成骨分化的影響[A];全國自身免疫性疾病專題研討會暨第十一次全國風(fēng)濕病學(xué)學(xué)術(shù)年會論文匯編[C];2006年

6 宋忠臣;束蓉;董家辰;李松;;低氧對牙周膜成纖維細胞增殖和成骨分化的影響[A];第十次全國牙周病學(xué)學(xué)術(shù)會議論文摘要匯編[C];2014年

7 董家辰;束蓉;宋忠臣;;炎癥微環(huán)境對人牙周膜成纖維細胞增殖與成骨分化的影響[A];第十次全國牙周病學(xué)學(xué)術(shù)會議論文摘要匯編[C];2014年

8 孫楠;楊力;張振;張樺;陳宏;蔡德鴻;;晚期氧化蛋白產(chǎn)物對大鼠骨髓間充質(zhì)干細胞增殖及向成骨分化的影響[A];中華醫(yī)學(xué)會第十二次全國內(nèi)分泌學(xué)學(xué)術(shù)會議論文匯編[C];2013年

9 馬雪;孟靜茹;賈敏;王寧;胡靜;周穎;羅曉星;;胰高血糖素樣肽-1類似物對大鼠骨髓間充質(zhì)干細胞增殖與成骨分化的影響[A];第十一屆全國青年藥學(xué)工作者最新科研成果交流會論文集[C];2012年

10 林和敏;;成骨分化的人臍血間充質(zhì)干細胞免疫原性研究[A];2013年全國激光醫(yī)學(xué)學(xué)術(shù)聯(lián)合會議暨2013年浙江省醫(yī)學(xué)會整形美容學(xué)術(shù)年會論文匯編[C];2013年

相關(guān)博士學(xué)位論文 前10條

1 龔逸明;microRNAs調(diào)控Satb2介導(dǎo)的骨髓基質(zhì)干細胞成骨分化的作用研究[D];復(fù)旦大學(xué);2014年

2 李松濤;EphB信號在軸向仿生壓應(yīng)力調(diào)控MSC成骨分化中的作用和機制研究[D];第三軍醫(yī)大學(xué);2015年

3 方文;純鈦表面WNT信號通路調(diào)控BMSCs成骨分化的機制研究[D];浙江大學(xué);2014年

4 劉世宇;移植外源性MSCs持久恢復(fù)宿主MSCs功能的機制研究[D];第四軍醫(yī)大學(xué);2015年

5 張莉;Fgfr2~(S252W/+)小鼠骨量、骨結(jié)構(gòu)特性及BMSCs成骨分化調(diào)控機制的研究[D];第三軍醫(yī)大學(xué);2015年

6 劉祥舟;機械牽伸通過Wnt5a、Wnt5b/JNK信號通路促進大鼠肌腱干細胞成骨分化[D];第三軍醫(yī)大學(xué);2015年

7 宋明宇;電磁場對地塞米松干預(yù)下骨髓間充質(zhì)干細胞增殖分化的效應(yīng)及機制研究[D];華中科技大學(xué);2015年

8 賈杰;MiR-17-5p調(diào)控非創(chuàng)傷性股骨頭壞死骨髓基質(zhì)干細胞成骨分化與增殖的相關(guān)研究[D];華中科技大學(xué);2015年

9 楊勇;電磁場促進骨髓間充質(zhì)干細胞成骨分化及初步機制[D];華中科技大學(xué);2010年

10 王琪;雌激素受體-α在骨髓間充質(zhì)干細胞中的表達及對成骨分化影響的研究[D];第四軍醫(yī)大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 吳小瑩;腦源性神經(jīng)營養(yǎng)因子對人脂肪干細胞增殖與成骨分化的作用[D];北京協(xié)和醫(yī)學(xué)院;2015年

2 秦子順;淫羊藿苷對人牙周膜干細胞的增殖和成骨分化的影響[D];蘭州大學(xué);2015年

3 劉可;miR-106bE靶向調(diào)控BMP2參與間充質(zhì)干細胞成骨分化與體內(nèi)骨形成[D];蘇州大學(xué);2015年

4 付雪杰;MSCs與去分化MSCs在成骨分化過程中免疫原性上調(diào)的研究[D];蘇州大學(xué);2015年

5 崔陽陽;蛋白激酶C在小鼠骨髓間充質(zhì)干細胞成骨分化中的作用研究[D];新鄉(xiāng)醫(yī)學(xué)院;2015年

6 嚴一杰;體外誘導(dǎo)人腎間質(zhì)成纖維細胞成骨分化的實驗研究[D];廣西醫(yī)科大學(xué);2015年

7 高羽萱;直流微電場對種植體周骨髓間充質(zhì)干細胞遷移和成骨影響的研究[D];中國人民解放軍醫(yī)學(xué)院;2015年

8 郭中豪;甲狀旁腺激素對大鼠骨髓間充質(zhì)干細胞成骨分化的影響[D];山西醫(yī)科大學(xué);2015年

9 李慧垠;高脂環(huán)境下大鼠BMSCs成骨分化過程中Wnt通路相關(guān)因子的表達變化[D];山東大學(xué);2015年

10 聶曉萌;MicroRNA靶向Id1對BMSCs成骨分化的影響[D];山東大學(xué);2015年

，

本文編號：1988735