本文選題：終板軟骨細胞　切入點：間歇性循環(huán)牽張力　出處：《皖南醫(yī)學院》2017年碩士論文

【摘要】：目的:體外培養(yǎng)建立大鼠終板軟骨細胞張力誘導炎癥模型,探討NF-κB信號通路與P120-catenin在張力誘導終板軟骨細胞炎癥中的調(diào)控機制。方法:無菌條件下取出大鼠的腰椎終板軟骨,膠原酶消化法提取終板軟骨細胞,細胞培養(yǎng)至二代后,采用FX-5000細胞應變加載系統(tǒng)體外誘導構建終板軟骨細胞張力載荷下的炎癥模型;采用特異性抑制劑Bay117082調(diào)控NF-κB信號通路;通過基因轉染過表達P120-catenin基因。實時RT-PCR檢測加力前后軟骨炎癥因子MMP3,MMP9,MMP13,COX-2,iNOS等基因的表達變化;AlamarBlue法檢測終板軟骨細胞增殖率、流式細胞術檢測細胞凋亡,應用鬼筆環(huán)肽染色檢測細胞加力前后骨架的變化;免疫熒光法檢測P120-catenin在終板軟骨細胞胞中的表達與定位;核質分離及蛋白免疫印跡檢測胞核與胞質中NF-κB信號通路相關蛋白P65、p-P65、IkBα、p-IkBα表達變化;Dual-Luciferase報告基因檢測NF-κB信號通路激活狀態(tài)。結果:張力加載會誘導終板軟骨細胞發(fā)生炎癥性反應,相關炎癥基因的表達上調(diào)。張力刺激不影響終板軟骨細胞的增殖,不會誘導細胞的發(fā)生凋亡,但會使軟骨細胞骨架發(fā)生一定程度的形態(tài)改變。隨著張力加載的進行,終板軟骨細胞中NF-κB信號通路激活,加力時間延長后炎癥相關因子的表達明顯增高;抑制NF-κB信號通路后,相關軟骨細胞炎癥因子表達降低,終板軟骨細胞炎性癥狀減輕。張力載荷下終板軟骨細胞中P120-catenin在基因和蛋白水平的表達均明顯降低,質粒轉染過表達P120-catenin后,軟骨細胞中P120-catenin在基因和蛋白水平表達顯著增高,張力刺激NF-κB信號通路激活被抑制,張力誘導的終板軟骨細胞炎性癥狀明顯減輕。結論:張力載荷通過誘導NF-κB信號通路的激活而導致終板軟骨細胞炎癥反應;這種調(diào)控是由于張力刺激下調(diào)軟骨細胞質內(nèi)的P120-catenin,減弱了P120-catenin對NF-κB信號通路的抑制作用,最終導致終板軟骨細胞的炎癥反應。調(diào)控NF-κB信號通路及P120-catenin的表達能夠在很大程度上減輕終板軟骨的炎性癥狀。
[Abstract]:Objective: to establish the in vitro tension of rat endplate chondrocyte induced inflammation model of NF- B signaling pathway and P120-catenin in tension induced regulation mechanism of endplate chondrocyte inflammation in rats. Methods: the lumbar endplate cartilage removed under sterile conditions, extraction of endplate cartilage cells by collagenase digestion, the cells were cultured for two generations, the by constructing inflammation model of endplate chondrocytes tension loading FX-5000 cell strain loading system in vitro; the specific inhibitor of Bay117082 regulation of NF- B signaling pathway by gene transfection; expression of P120-catenin gene. After real-time RT-PCR for the detection of inflammatory factors MMP3 cartilage afterburner MMP9, MMP13, COX-2, and the expression change of iNOS gene; detection of endplate cartilage cell proliferation AlamarBlue assay, flow cytometry, cell skeleton change detected before and after the application of phalloidin staining afterburner; The expression and localization of immunofluorescence was used to detect P120-catenin in the cytoplasm of endplate chondrocytes; NF- kappa B signaling pathway related protein P65, cell separation and detection of nuclear protein nuclear and cytoplasmic p-P65, IkB alpha, alpha p-IkB expression; Dual-Luciferase reporter gene detection of NF- kappa B signaling pathway activation. Results: tension the loading will induce inflammatory reaction of endplate cartilage cells, upregulation of inflammatory gene expression. The tension did not affect the endplate cartilage cell proliferation, apoptosis does not induce cells, but the change will make a certain degree of cartilage cytoskeleton morphology. With tension loading, NF- kappa B signaling pathway of endplate chondrocytes. After long time, boosting of inflammatory cytokines expression was significantly increased; the inhibition of NF- B signaling pathway and related inflammatory factors decreased expression of cartilage cells, cartilage endplate cells inflammatory symptoms Reduce the expression of P120-catenin. Endplate chondrocytes tension loads in gene and protein levels were significantly decreased after overexpression of P120-catenin plasmid transfection, expression of P120-catenin in cartilage cells in gene and protein levels increased significantly, tension stimulation of NF- B signaling pathway activation is inhibited, the tension induced by endplate chondrocyte inflammatory symptoms significantly reduced. Conclusion: the result of endplate cartilage cell inflammatory response induced by activation of NF- tension load B pathway; this regulation is stimulated by cartilage due to tension in the cytoplasm of P120-catenin, weaken the inhibitory effect of P120-catenin on NF- B signaling pathway, resulting in inflammation of endplate cartilage cells. The expression of B signaling pathway and regulation of NF- P120-catenin can reduce the endplate cartilage largely inflammatory symptoms.

【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R681.53

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本文編號：1681485