Hedgehog通路在佐剂性关节炎大鼠关节软骨中的表达及部分功能研究

发布时间：2018-05-12 07:55

本文选题：Hedgehog信号通路 + 佐剂性关节炎　；参考：《安徽医科大学》2015年硕士论文

【摘要】：目的探讨Hedgehog(Hh)通路在佐剂性关节炎(AIA)大鼠软骨中的表达及意义;及研究Hh通路阻断剂环巴胺对AIA大鼠关节软骨细胞增殖凋亡的影响及抗凋亡机制。方法弗氏完全佐剂诱导AIA大鼠模型;免疫组织化学(SP)法观察Hh信号通路相关分子(Shh,Ptch1,Smo,Gli1)在AIA大鼠关节软骨中的蛋白表达情况;实时荧光定量PCR的方法检测Shh,Ptch1,Smo,Gli1,II型胶原(COII)和蛋白聚糖(aggrecan)的m RNA表达水平;并且把Hh信号通路相关分子的m RNA水平与AIA大鼠关节软骨损伤程度(OARSI评分,COII和aggrecan m RNA表达水平)进行相关性分析。胰酶-胶原酶法分离培养关节软骨细胞;环巴胺体外给药处理AIA大鼠软骨细胞;MTT法检测细胞增殖;DNA电泳、Hoechst染色、Annexin V-FITC/PI双染检测细胞凋亡;RT-PCR检测Shh,Gli1,Ptch1,Bcl-2,Bax和Caspase-3 m RNA表达。结果1.Hh通路在AIA大鼠软骨中的表达及意义AIA大鼠造模成功的基础上,Shh,Ptch1,Smo,Gli1的蛋白和基因水平在AIA大鼠中的表达均明显高于正常组;AIA大鼠软骨组织中的COII和aggrecan的m RNA表达水平则显著降低;AIA大鼠中Shh,Ptch1,Smo,Gli1 m RNA的表达水平与OARSI评分明显正相关,与关节软骨组织中COII,aggrecan m RNA表达水平明显负相关;2.环巴胺对佐剂性关节炎大鼠关节软骨细胞增殖凋亡的影响及其抗凋亡机制环巴胺(0.08,0.4,2,10,50μmol/L)剂量依赖性地提高AIA大鼠软骨细胞增殖;AIA组可见凋亡细胞DNA梯状条带,环巴胺(0.4,2,10μmol/L)给药组的梯状条带明显减少;AIA组存在核碎裂与染色质固缩,环巴胺给药组均匀蓝色荧光的细胞数量增多;流式分析结果表明AIA大鼠软骨细胞凋亡率显著升高,环巴胺明显减少细胞凋亡率;与正常组相比,AIA大鼠软骨细胞中Hh通路相关分子(Shh,Ptch1,Gli1)m RNA表达显著升高,抗凋亡基因Bcl-2 m RNA表达显著下降,而促凋亡基因Bax,Caspase-3 m RNA表达明显升高,环巴胺体外给药能抑制Hh通路过度活化,提高Bcl-2 m RNA表达,并降低Bax,Caspase-3 m RNA表达。结论1.Hh信号通路在AIA大鼠关节软骨中明显异常活化,并密切参与AIA大鼠关节软骨损伤的病理过程,提示其有望成为类风湿关节炎治疗的新靶点;2.环巴胺体外给药能促进AIA大鼠软骨细胞的增殖,并抑制AIA大鼠软骨细胞的过度凋亡,该抗凋亡作用和调节Bcl-2,Bax和Caspase-3表达密切相关,提示干预软骨细胞Hh通路对保护类风湿关节炎关节软骨具有潜在的治疗意义。
[Abstract]:Objective to investigate the expression and significance of Hedgehog Hh pathway in the cartilage of rats with adjuvant arthritis (AIA), and to study the effect of cyclobaramine, an Hh pathway blocker, on proliferation and apoptosis of articular chondrocytes in AIA rats. Methods the AIA rat model was induced by Freund's complete adjuvant, and the expression of Hh signal pathway related molecule Smog Gli1 in the articular cartilage of AIA rats was observed by immunohistochemistry. The expression of m RNA was detected by real-time fluorescence quantitative PCR. The correlation between the level of m RNA related to Hh signaling pathway and the degree of articular cartilage injury in AIA rats was analyzed. Cultured articular chondrocytes were isolated and cultured by trypsin collagenase method, AIA chondrocytes were treated with cyclobaramine in vitro, cell proliferation DNA electrophoresis and Annexin V-FITC/PI double staining were used to detect cell apoptosis and RT-PCR was used to detect the expression of Bcl-2Bcl-2Bcl-2Bax and Caspase-3 m RNA in AIA rat chondrocytes. Results the expression of 1.Hh pathway in cartilage of AIA rats and its significance; on the basis of successful modeling of AIA rats, the expression of protein and gene of AIA Ptch1SmoGli1 in AIA rats was significantly higher than that of COII and aggrecan in chondrocytes of normal rats. There was a significant positive correlation between the OARSI score and the expression of Smog Gli1 m RNA in AIA rats. There was a significant negative correlation between the expression of COIIGGREM RNA in articular cartilage and the expression of COIIGGREM RNA in articular cartilage. Effects of cyclobaramine on proliferation and apoptosis of articular chondrocytes in rats with adjuvant arthritis The ladder band of cyclophosphamide (0.4 渭 mol / L) group significantly reduced nuclear fragmentation and chromatin pyknosis, increased the number of uniform blue fluorescence cells in AIA group, and increased the apoptotic rate of chondrocytes in AIA rats. Compared with the normal control group, the expression of Hh pathway related molecule RNA in chondrocytes was significantly increased, the expression of anti-apoptotic gene Bcl-2 m RNA was significantly decreased, while the expression of Baxon Caspase-3 m RNA was significantly increased. Cyclopramine could inhibit the overexpression of Hh pathway, increase the expression of Bcl-2 m RNA and decrease the expression of Caspase-3 m RNA in vitro. Conclusion 1.Hh signaling pathway is significantly abnormal in the articular cartilage of AIA rats and is closely involved in the pathological process of articular cartilage injury in AIA rats, suggesting that it may be a new target for the treatment of rheumatoid arthritis. Cyclopramine could promote the proliferation of chondrocytes in AIA rats in vitro and inhibit the excessive apoptosis of chondrocytes in AIA rats. The anti-apoptotic effect was closely related to the regulation of Bcl-2 Bax and Caspase-3 expression. It is suggested that the intervention of Hh pathway in chondrocytes may be of potential therapeutic significance in protecting the articular cartilage of rheumatoid arthritis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3

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