本文選題：骨性關節(jié)炎 + miR-145��； 參考：《天津醫(yī)科大學》2015年博士論文

【摘要】：研究目的探討mi R-145在原發(fā)性膝關節(jié)骨性關節(jié)炎軟骨中的表達及其對正常軟骨細胞的生物學作用,從而為探討其在骨性關節(jié)炎中的作用機制奠定基礎。研究方法收集原發(fā)性膝關節(jié)骨性關節(jié)炎20例和正常膝關節(jié)的關節(jié)軟骨10例,對軟骨進行細胞培養(yǎng),并根據(jù)改良的Mankin評分標準對骨性關節(jié)炎關節(jié)軟骨進行分組。應用免疫組化的方法檢測mi R-145在各組軟骨組織中的表達;使用RT-PCR方法檢測各組軟骨組織中mi R-145的表達,應用RT-PCR方法檢測軟骨細胞中MMP13、X型膠原、RNAK、堿性磷酸酶m RNA的表達,western blot檢測軟骨細胞中MMP13、X型膠原、RANK、堿性磷酸酶的蛋白表達;所有數(shù)據(jù)均應用SPSS 19.0軟件進行統(tǒng)計學分析。通過生物信息分析技術分析軟骨細胞中mi R-145的靶向調(diào)控基因表達差異,尋找軟骨與mi R-145共表達基因,并驗證。通過Lipofectamine?2000試劑將其轉(zhuǎn)染軟骨細胞,通過RT-PCR檢測軟骨細胞中相關基因的表達量,westernblot檢測軟骨細胞中相關蛋白的表達量。通過干擾TNFRSF11B,探索其對mi R-145抑制的軟骨細胞膠原蛋白表達的影響。研究結(jié)果Mi R-145在正常膝關節(jié)和原發(fā)性膝關節(jié)骨性關節(jié)炎的關節(jié)軟骨中均有表達,其主要位于軟骨細胞的胞漿中,RT-PCR結(jié)果顯示mi R-145的表達在骨性關節(jié)炎軟骨組織中明顯低于正常膝關節(jié)的關節(jié)軟骨(P0.05),western blot的結(jié)果同樣提示原發(fā)性骨性關節(jié)炎軟骨細胞中mi R-145蛋白含量均顯著低于正常膝關節(jié)軟骨(P0.05),而且隨著骨性關節(jié)炎嚴重程度的提高,mi R-145的表達、蛋白含量逐步降低,在重度骨性關節(jié)炎軟骨中這些差異都具有統(tǒng)計學意義(P0.05)。為了進一步探討mi R-145對正常軟骨細胞生物學的影響,我們成功將其轉(zhuǎn)染至培養(yǎng)的軟骨細胞,RT-PCR和western blot對軟骨細胞進行檢測分析,結(jié)果顯示:MMP13、Collagen X膠原纖維蛋白受到抑制;大劑量時(100n M),RANK蛋白表達受到抑制,對ALP的作用不明顯;但上述抑制并不依賴于mi R-145的濃度。對GSE17785表達普芯片進行分析,骨關節(jié)炎相關基因為732個,mi R-145相關基因有192個,同時與軟骨相關和mi R-145相關的基因有13個;在mi R-145過表達質(zhì)粒轉(zhuǎn)染軟骨細胞后,對關鍵基因蛋白TNFRSF11B進行檢測,結(jié)果表明,隨著mi R-145濃度增加,TNFRSF11B蛋白表達降低。正常關節(jié)軟骨與不同退變程度的骨關節(jié)炎(輕度、中度及重度),其軟骨組織中TNFRSF11B表達隨著退變程度加重,表達水平增高。方差分析顯示,TNFRSF11B在正常組織中表達最低,在重度退變軟骨中呈高表達,與其余三組的差異有統(tǒng)計學意義(P0.01),而在輕度和重度退變的組織中表達差異無統(tǒng)計學意義(P0.05)。關節(jié)軟骨細胞經(jīng)TNF-a誘導處理,檢測其中TNFRSF11B基因表達,驗證軟骨細胞中TNFRSF11B基因變化,結(jié)果顯示,TNFRSF11B在骨關節(jié)炎軟骨細胞中高表達,在正常軟骨細胞中低表達。對軟骨細胞TNFRSF11B進行干擾,并進行mi R-145過表達質(zhì)粒轉(zhuǎn)染,檢測細胞中與軟骨相關的蛋白的表達,研究結(jié)果表明,MMP13和ALP蛋白變化趨勢不明顯,RANK蛋白變化明顯,collagen X和TNFRSF11B蛋白明顯增加。研究結(jié)論1、原發(fā)性膝關節(jié)骨性關節(jié)炎的軟骨中mi R-145的表達較正常軟骨顯著降低,并且軟骨退變程度越重,mi R-145表達亦越低;2、mi R-145可以抑制軟骨細胞MMP13、X型膠原、RANK的表達;3、mi R-145靶向調(diào)控TNFRSF11B;4、TNFRSF11B在關節(jié)軟骨中高表達;5、干擾TNFRSF11B可以rescue mi R-145抑制的軟骨細胞膠原蛋白表達。
[Abstract]:Objective to investigate the expression of MI R-145 in the cartilage of primary osteoarthritis of the knee and its biological effect on normal cartilage cells, so as to explore the mechanism of its action in osteoarthritis. The research method collected 10 cases of primary osteoarthritis of the knee joint and the articular cartilage of the normal knee joint, and the cartilage of 20 cases and the cartilage of the normal knee joint. Cell culture was carried out and the articular cartilage of osteoarthritis was grouped according to the improved Mankin scoring standard. The expression of MI R-145 in cartilage tissue was detected by immunohistochemical method. The expression of MI R-145 in cartilage tissue was detected by RT-PCR method. RT-PCR method was used to detect MMP13, X collagen and RNAK in cartilage cells. The expression of alkaline phosphatase m RNA, Western blot was used to detect the protein expression of MMP13, X collagen, RANK, alkaline phosphatase in cartilage cells. All data were statistically analyzed with SPSS 19 software. The expression of MI R-145 in cartilage cells was analyzed by bioinformatics analysis technique, and the co expression of cartilage and mi R-145 was found. Gene, and verified. Transfection with Lipofectamine 2000 reagent to chondrocytes, detecting the expression of related genes in cartilage cells by RT-PCR, Westernblot detection of the expression of related proteins in cartilage cells. The effect of TNFRSF11B on the expression of collagen in cartilage cells inhibited by Mi R-145 was explored. The result of the study was Mi R-145. In the articular cartilage of normal knee and primary osteoarthritis of the knee, it is mainly located in the cytoplasm of chondrocytes. RT-PCR results show that the expression of MI R-145 in osteoarthritis cartilage is significantly lower than that of the articular cartilage of normal knee joint (P0.05). The result of Western blot also suggests the primary bone joint. The content of MI R-145 protein in inflammatory chondrocytes was significantly lower than that of normal articular cartilage (P0.05), and with the increase of osteoarthritis severity, the expression of MI R-145 and the protein content gradually decreased, and these differences in severe osteoarthritis cartilage were statistically significant (P0.05). In order to further explore mi R-145 to normal cartilage thin. The effects of cell biology were successfully transfected into cultured chondrocytes, and RT-PCR and Western blot were detected and analyzed. The results showed that MMP13, Collagen X collagen fibrin was suppressed; the expression of RANK protein was inhibited in high dose (100N M), and the effect on ALP was not obvious; but the inhibition was not dependent on MI R-145. Analysis of GSE17785 expression, 732 genes related to osteoarthritis, 192 mi R-145 related genes, and 13 genes related to cartilage and MI R-145; the key gene protein TNFRSF11B was detected after MI R-145 overexpressed plasmids transfected to chondrocytes, and the results showed that the concentration of MI R-145 increased. The expression of TNFRSF11B protein decreased. Normal articular cartilage and osteoarthritis of different degrees of degeneration (mild, moderate and severe), the expression of TNFRSF11B in the cartilage tissue increased with the degree of degeneration, and the expression level increased. The analysis of variance analysis showed that the expression of TNFRSF11B was the lowest in normal tissue, high expression in severe degeneration cartilage, and the other three groups. The difference was statistically significant (P0.01), but there was no significant difference in expression in mild and severe degeneration tissues (P0.05). The expression of TNFRSF11B gene was detected by TNF-a induction treatment in articular chondrocytes, and the change of TNFRSF11B gene in cartilage cells was verified. The results showed that TNFRSF11B was highly expressed in osteoarthritis cartilage cells, and in normal soft tissue. Low expression in bone cells. Interference with cartilage cell TNFRSF11B and transfection of MI R-145 overexpressed plasmid to detect the expression of cartilage related proteins in cells. The results showed that the change trend of MMP13 and ALP protein was not obvious, the changes of RANK protein were obvious, collagen X and TNFRSF11B protein were obviously increased. Conclusion 1, primary knee clearance The expression of MI R-145 in cartilaginous cartilage of osteoarthrosis was significantly lower than that of normal cartilage, and the higher the degree of cartilage degeneration, the lower the expression of MI R-145; 2, MI R-145 could inhibit the expression of MMP13, X type collagen and RANK in cartilage cells; 3, MI R-145 targeted TNFRSF11B; 4, high expression in articular cartilage; 5 R-145 inhibits the expression of collagen in chondrocytes.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R684.3

【共引文獻】

相關期刊論文 前9條

1 侯良磊;楊德業(yè);;微小RNA與高血壓的研究進展[J];國際心血管病雜志;2014年01期

2 雷桅;稅曉容;陳燦;;microRNA在冠心病發(fā)生機制中的作用及其臨床應用[J];廣東醫(yī)學;2014年17期

3 陳培芬;邱智輝;黃國華;張香梅;彭武建;曾輝;賴文巖;;Anti-miR-145促進人氣道平滑肌細胞增殖及骨橋蛋白合成[J];南方醫(yī)科大學學報;2015年07期

4 賈倩;何昆侖;;心血管疾病的生物標志物研究進展[J];標記免疫分析與臨床;2015年10期

5 孫嘉偉;劉鳳霞;王似雄;;MicroRNA-145在各類腫瘤的表達水平[J];農(nóng)墾醫(yī)學;2015年03期

6 晏蕭;姜睿;;非編碼RNAs與勃起功能障礙關系研究進展[J];中華男科學雜志;2016年02期

7 朱明燕;莫中成;曾高峰;;血管平滑肌細胞增殖相關microRNA的研究進展[J];實用醫(yī)學雜志;2013年21期

8 郝理;游盛中;馬素華;張東川;羅斌;權力;;miR-143、miR-145及血管緊張素II在人冠狀動脈粥樣硬化斑塊中的表達[J];熱帶醫(yī)學雜志;2014年04期

9 徐冉;鐘久昌;;微小RNA和血管緊張素轉(zhuǎn)換酶2/血管緊張素(1~7)信號與高血壓[J];上海醫(yī)學;2015年04期

相關會議論文 前1條

1 趙施皓;楊智偉;鄭煦f，

本文編號：1850382