MiR-145在原发性膝关节骨性关节炎软骨中的表达及其意义

发布时间：2018-05-06 02:27

本文选题：骨性关节炎 + miR-145　；参考：《天津医科大学》2015年博士论文

【摘要】：研究目的探讨mi R-145在原发性膝关节骨性关节炎软骨中的表达及其对正常软骨细胞的生物学作用,从而为探讨其在骨性关节炎中的作用机制奠定基础。研究方法收集原发性膝关节骨性关节炎20例和正常膝关节的关节软骨10例,对软骨进行细胞培养,并根据改良的Mankin评分标准对骨性关节炎关节软骨进行分组。应用免疫组化的方法检测mi R-145在各组软骨组织中的表达;使用RT-PCR方法检测各组软骨组织中mi R-145的表达,应用RT-PCR方法检测软骨细胞中MMP13、X型胶原、RNAK、碱性磷酸酶m RNA的表达,western blot检测软骨细胞中MMP13、X型胶原、RANK、碱性磷酸酶的蛋白表达;所有数据均应用SPSS 19.0软件进行统计学分析。通过生物信息分析技术分析软骨细胞中mi R-145的靶向调控基因表达差异,寻找软骨与mi R-145共表达基因,并验证。通过Lipofectamine?2000试剂将其转染软骨细胞,通过RT-PCR检测软骨细胞中相关基因的表达量,westernblot检测软骨细胞中相关蛋白的表达量。通过干扰TNFRSF11B,探索其对mi R-145抑制的软骨细胞胶原蛋白表达的影响。研究结果Mi R-145在正常膝关节和原发性膝关节骨性关节炎的关节软骨中均有表达,其主要位于软骨细胞的胞浆中,RT-PCR结果显示mi R-145的表达在骨性关节炎软骨组织中明显低于正常膝关节的关节软骨(P0.05),western blot的结果同样提示原发性骨性关节炎软骨细胞中mi R-145蛋白含量均显著低于正常膝关节软骨(P0.05),而且随着骨性关节炎严重程度的提高,mi R-145的表达、蛋白含量逐步降低,在重度骨性关节炎软骨中这些差异都具有统计学意义(P0.05)。为了进一步探讨mi R-145对正常软骨细胞生物学的影响,我们成功将其转染至培养的软骨细胞,RT-PCR和western blot对软骨细胞进行检测分析,结果显示:MMP13、Collagen X胶原纤维蛋白受到抑制;大剂量时(100n M),RANK蛋白表达受到抑制,对ALP的作用不明显;但上述抑制并不依赖于mi R-145的浓度。对GSE17785表达普芯片进行分析,骨关节炎相关基因为732个,mi R-145相关基因有192个,同时与软骨相关和mi R-145相关的基因有13个;在mi R-145过表达质粒转染软骨细胞后,对关键基因蛋白TNFRSF11B进行检测,结果表明,随着mi R-145浓度增加,TNFRSF11B蛋白表达降低。正常关节软骨与不同退变程度的骨关节炎(轻度、中度及重度),其软骨组织中TNFRSF11B表达随着退变程度加重,表达水平增高。方差分析显示,TNFRSF11B在正常组织中表达最低,在重度退变软骨中呈高表达,与其余三组的差异有统计学意义(P0.01),而在轻度和重度退变的组织中表达差异无统计学意义(P0.05)。关节软骨细胞经TNF-a诱导处理,检测其中TNFRSF11B基因表达,验证软骨细胞中TNFRSF11B基因变化,结果显示,TNFRSF11B在骨关节炎软骨细胞中高表达,在正常软骨细胞中低表达。对软骨细胞TNFRSF11B进行干扰,并进行mi R-145过表达质粒转染,检测细胞中与软骨相关的蛋白的表达,研究结果表明,MMP13和ALP蛋白变化趋势不明显,RANK蛋白变化明显,collagen X和TNFRSF11B蛋白明显增加。研究结论1、原发性膝关节骨性关节炎的软骨中mi R-145的表达较正常软骨显著降低,并且软骨退变程度越重,mi R-145表达亦越低;2、mi R-145可以抑制软骨细胞MMP13、X型胶原、RANK的表达;3、mi R-145靶向调控TNFRSF11B;4、TNFRSF11B在关节软骨中高表达;5、干扰TNFRSF11B可以rescue mi R-145抑制的软骨细胞胶原蛋白表达。
[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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R684.3

【共引文献】