本文選題：Hedgehog信號通路 + 佐劑性關(guān)節(jié)炎　； 參考：《安徽醫(yī)科大學》2015年碩士論文

【摘要】：目的探討Hedgehog(Hh)通路在佐劑性關(guān)節(jié)炎(AIA)大鼠軟骨中的表達及意義;及研究Hh通路阻斷劑環(huán)巴胺對AIA大鼠關(guān)節(jié)軟骨細胞增殖凋亡的影響及抗凋亡機制。方法弗氏完全佐劑誘導AIA大鼠模型;免疫組織化學(SP)法觀察Hh信號通路相關(guān)分子(Shh,Ptch1,Smo,Gli1)在AIA大鼠關(guān)節(jié)軟骨中的蛋白表達情況;實時熒光定量PCR的方法檢測Shh,Ptch1,Smo,Gli1,II型膠原(COII)和蛋白聚糖(aggrecan)的m RNA表達水平;并且把Hh信號通路相關(guān)分子的m RNA水平與AIA大鼠關(guān)節(jié)軟骨損傷程度(OARSI評分,COII和aggrecan m RNA表達水平)進行相關(guān)性分析。胰酶-膠原酶法分離培養(yǎng)關(guān)節(jié)軟骨細胞;環(huán)巴胺體外給藥處理AIA大鼠軟骨細胞;MTT法檢測細胞增殖;DNA電泳、Hoechst染色、Annexin V-FITC/PI雙染檢測細胞凋亡;RT-PCR檢測Shh,Gli1,Ptch1,Bcl-2,Bax和Caspase-3 m RNA表達。結(jié)果1.Hh通路在AIA大鼠軟骨中的表達及意義AIA大鼠造模成功的基礎(chǔ)上,Shh,Ptch1,Smo,Gli1的蛋白和基因水平在AIA大鼠中的表達均明顯高于正常組;AIA大鼠軟骨組織中的COII和aggrecan的m RNA表達水平則顯著降低;AIA大鼠中Shh,Ptch1,Smo,Gli1 m RNA的表達水平與OARSI評分明顯正相關(guān),與關(guān)節(jié)軟骨組織中COII,aggrecan m RNA表達水平明顯負相關(guān);2.環(huán)巴胺對佐劑性關(guān)節(jié)炎大鼠關(guān)節(jié)軟骨細胞增殖凋亡的影響及其抗凋亡機制環(huán)巴胺(0.08,0.4,2,10,50μmol/L)劑量依賴性地提高AIA大鼠軟骨細胞增殖;AIA組可見凋亡細胞DNA梯狀條帶,環(huán)巴胺(0.4,2,10μmol/L)給藥組的梯狀條帶明顯減少;AIA組存在核碎裂與染色質(zhì)固縮,環(huán)巴胺給藥組均勻藍色熒光的細胞數(shù)量增多;流式分析結(jié)果表明AIA大鼠軟骨細胞凋亡率顯著升高,環(huán)巴胺明顯減少細胞凋亡率;與正常組相比,AIA大鼠軟骨細胞中Hh通路相關(guān)分子(Shh,Ptch1,Gli1)m RNA表達顯著升高,抗凋亡基因Bcl-2 m RNA表達顯著下降,而促凋亡基因Bax,Caspase-3 m RNA表達明顯升高,環(huán)巴胺體外給藥能抑制Hh通路過度活化,提高Bcl-2 m RNA表達,并降低Bax,Caspase-3 m RNA表達。結(jié)論1.Hh信號通路在AIA大鼠關(guān)節(jié)軟骨中明顯異�；罨�,并密切參與AIA大鼠關(guān)節(jié)軟骨損傷的病理過程,提示其有望成為類風濕關(guān)節(jié)炎治療的新靶點;2.環(huán)巴胺體外給藥能促進AIA大鼠軟骨細胞的增殖,并抑制AIA大鼠軟骨細胞的過度凋亡,該抗凋亡作用和調(diào)節(jié)Bcl-2,Bax和Caspase-3表達密切相關(guān),提示干預軟骨細胞Hh通路對保護類風濕關(guān)節(jié)炎關(guān)節(jié)軟骨具有潛在的治療意義。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R684.3

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