發(fā)布時(shí)間：2018-05-06 02:27

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

【摘要】：研究目的探討mi R-145在原發(fā)性膝關(guān)節(jié)骨性關(guān)節(jié)炎軟骨中的表達(dá)及其對(duì)正常軟骨細(xì)胞的生物學(xué)作用,從而為探討其在骨性關(guān)節(jié)炎中的作用機(jī)制奠定基礎(chǔ)。研究方法收集原發(fā)性膝關(guān)節(jié)骨性關(guān)節(jié)炎20例和正常膝關(guān)節(jié)的關(guān)節(jié)軟骨10例,對(duì)軟骨進(jìn)行細(xì)胞培養(yǎng),并根據(jù)改良的Mankin評(píng)分標(biāo)準(zhǔn)對(duì)骨性關(guān)節(jié)炎關(guān)節(jié)軟骨進(jìn)行分組。應(yīng)用免疫組化的方法檢測(cè)mi R-145在各組軟骨組織中的表達(dá);使用RT-PCR方法檢測(cè)各組軟骨組織中mi R-145的表達(dá),應(yīng)用RT-PCR方法檢測(cè)軟骨細(xì)胞中MMP13、X型膠原、RNAK、堿性磷酸酶m RNA的表達(dá),western blot檢測(cè)軟骨細(xì)胞中MMP13、X型膠原、RANK、堿性磷酸酶的蛋白表達(dá);所有數(shù)據(jù)均應(yīng)用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。通過(guò)生物信息分析技術(shù)分析軟骨細(xì)胞中mi R-145的靶向調(diào)控基因表達(dá)差異,尋找軟骨與mi R-145共表達(dá)基因,并驗(yàn)證。通過(guò)Lipofectamine?2000試劑將其轉(zhuǎn)染軟骨細(xì)胞,通過(guò)RT-PCR檢測(cè)軟骨細(xì)胞中相關(guān)基因的表達(dá)量,westernblot檢測(cè)軟骨細(xì)胞中相關(guān)蛋白的表達(dá)量。通過(guò)干擾TNFRSF11B,探索其對(duì)mi R-145抑制的軟骨細(xì)胞膠原蛋白表達(dá)的影響。研究結(jié)果Mi R-145在正常膝關(guān)節(jié)和原發(fā)性膝關(guān)節(jié)骨性關(guān)節(jié)炎的關(guān)節(jié)軟骨中均有表達(dá),其主要位于軟骨細(xì)胞的胞漿中,RT-PCR結(jié)果顯示mi R-145的表達(dá)在骨性關(guān)節(jié)炎軟骨組織中明顯低于正常膝關(guān)節(jié)的關(guān)節(jié)軟骨(P0.05),western blot的結(jié)果同樣提示原發(fā)性骨性關(guān)節(jié)炎軟骨細(xì)胞中mi R-145蛋白含量均顯著低于正常膝關(guān)節(jié)軟骨(P0.05),而且隨著骨性關(guān)節(jié)炎嚴(yán)重程度的提高,mi R-145的表達(dá)、蛋白含量逐步降低,在重度骨性關(guān)節(jié)炎軟骨中這些差異都具有統(tǒng)計(jì)學(xué)意義(P0.05)。為了進(jìn)一步探討mi R-145對(duì)正常軟骨細(xì)胞生物學(xué)的影響,我們成功將其轉(zhuǎn)染至培養(yǎng)的軟骨細(xì)胞,RT-PCR和western blot對(duì)軟骨細(xì)胞進(jìn)行檢測(cè)分析,結(jié)果顯示:MMP13、Collagen X膠原纖維蛋白受到抑制;大劑量時(shí)(100n M),RANK蛋白表達(dá)受到抑制,對(duì)ALP的作用不明顯;但上述抑制并不依賴于mi R-145的濃度。對(duì)GSE17785表達(dá)普芯片進(jìn)行分析,骨關(guān)節(jié)炎相關(guān)基因?yàn)?32個(gè),mi R-145相關(guān)基因有192個(gè),同時(shí)與軟骨相關(guān)和mi R-145相關(guān)的基因有13個(gè);在mi R-145過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染軟骨細(xì)胞后,對(duì)關(guān)鍵基因蛋白TNFRSF11B進(jìn)行檢測(cè),結(jié)果表明,隨著mi R-145濃度增加,TNFRSF11B蛋白表達(dá)降低。正常關(guān)節(jié)軟骨與不同退變程度的骨關(guān)節(jié)炎(輕度、中度及重度),其軟骨組織中TNFRSF11B表達(dá)隨著退變程度加重,表達(dá)水平增高。方差分析顯示,TNFRSF11B在正常組織中表達(dá)最低,在重度退變軟骨中呈高表達(dá),與其余三組的差異有統(tǒng)計(jì)學(xué)意義(P0.01),而在輕度和重度退變的組織中表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。關(guān)節(jié)軟骨細(xì)胞經(jīng)TNF-a誘導(dǎo)處理,檢測(cè)其中TNFRSF11B基因表達(dá),驗(yàn)證軟骨細(xì)胞中TNFRSF11B基因變化,結(jié)果顯示,TNFRSF11B在骨關(guān)節(jié)炎軟骨細(xì)胞中高表達(dá),在正常軟骨細(xì)胞中低表達(dá)。對(duì)軟骨細(xì)胞TNFRSF11B進(jìn)行干擾,并進(jìn)行mi R-145過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染,檢測(cè)細(xì)胞中與軟骨相關(guān)的蛋白的表達(dá),研究結(jié)果表明,MMP13和ALP蛋白變化趨勢(shì)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(guān)節(jié)骨性關(guān)節(jié)炎的軟骨中mi R-145的表達(dá)較正常軟骨顯著降低,并且軟骨退變程度越重,mi R-145表達(dá)亦越低;2、mi R-145可以抑制軟骨細(xì)胞MMP13、X型膠原、RANK的表達(dá);3、mi R-145靶向調(diào)控TNFRSF11B;4、TNFRSF11B在關(guān)節(jié)軟骨中高表達(dá);5、干擾TNFRSF11B可以rescue mi R-145抑制的軟骨細(xì)胞膠原蛋白表達(dá)。
[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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R684.3

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