本文選題：關(guān)節(jié)軟骨細(xì)胞　切入點：生長分化因子5　出處：《新疆醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：觀察體外傳代過程中C57小鼠關(guān)節(jié)軟骨細(xì)胞去分化現(xiàn)象,GDF5作用于已去分化的軟骨細(xì)胞,使其再分化。兔軟骨細(xì)胞三維培養(yǎng),GDF5誘導(dǎo)后修復(fù)兔膝關(guān)節(jié)缺損,行檢測進(jìn)一步證明GDF5的作用。初步研究MiRNA-145在軟骨再分化中的作用機制。方法：新生C57小鼠膝關(guān)節(jié)分離獲取原代軟骨細(xì)胞傳代至P7代,對P1、P4代和GDF5誘導(dǎo)的P4代細(xì)胞從形態(tài)、增殖、基質(zhì)分泌以及基因和蛋白表達(dá)等方面采用顯微鏡觀察,CCK-8增殖曲線測定、細(xì)胞阿利辛藍(lán)染色、qRT-PCR及Western Blot進(jìn)行比較。將不同實驗組的兔軟骨細(xì)胞與PLGA/CHI絡(luò)合物支架結(jié)合7天后修復(fù)兔膝關(guān)節(jié)缺損,三月后取材,行HE、阿利辛藍(lán)、C012免疫組化染色等檢測。將MiRNA-145的模擬劑和抑制劑轉(zhuǎn)染入細(xì)胞,檢測胞內(nèi)SOX9水平。結(jié)果：C57小鼠及兔關(guān)節(jié)軟骨細(xì)胞傳代過程中形態(tài)發(fā)生變化；P4代細(xì)胞增殖能力較P1代稍減弱,P7代無明顯增殖；P1代胞外基質(zhì)分泌量高于P4代,而P4代經(jīng)GDF5誘導(dǎo)7天后表達(dá)量有所上升；P4代細(xì)胞相關(guān)特性分子(SOX9、Col Ⅱ和Aggrecan等)mRNA及蛋白表達(dá)水平較P1代下調(diào),經(jīng)GDF5誘導(dǎo)后表達(dá)水平回升；PLGA/CHI支架的細(xì)胞毒性低且細(xì)胞可均勻分布材料各層。P4細(xì)胞/材料GDF5誘導(dǎo)組修復(fù)缺損效果介于P1細(xì)胞/材料組和P4細(xì)胞/材料組之間；抑制MiRNA-145可增強SOX9的表達(dá),反之亦然。結(jié)論：體外培養(yǎng)的P4代軟骨細(xì)胞出現(xiàn)較明顯的去分化現(xiàn)象,經(jīng)一定濃度(300ng/ml)的GDF5單層誘導(dǎo)7天,SOX9、Col Ⅱ及Aggrecan含量回升,體內(nèi)細(xì)胞+PLGA/CHI支架復(fù)合物修復(fù)軟骨缺損的各項檢測結(jié)論一致。MiRNA-145對軟骨細(xì)胞去分化過程中SOX9的表達(dá)具有負(fù)調(diào)控作用。
[Abstract]:Objective: to observe the dedifferentiation of articular chondrocytes in C57 mice during passage in vitro and to observe the effect of GDF5 on dedifferentiated chondrocytes. The role of GDF5 in cartilage redifferentiation was studied. Methods: primary chondrocytes were isolated from the knee joint of newborn C57 mice and subcultured to P7 generation. The morphology and proliferation of P4 and P4 cells induced by GDF5 were observed. The proliferation curve of CCK-8 was observed by microscope in matrix secretion and gene and protein expression. The rabbit chondrocytes in different experimental groups were combined with PLGA/CHI complex scaffolds for 7 days to repair the knee joint defect, and the samples were obtained after March. The imitating agents and inhibitors of MiRNA-145 were transfected into the cells. Results during the passage of articular chondrocytes of mice and rabbits, the proliferative ability of P4 cells was slightly weaker than that of P1 generation. The secretion of extracellular matrix of P1 generation was higher than that of P4 generation. On the other hand, the expression of P4 cells increased after induction by GDF5 for 7 days. The mRNA and protein expression levels of P4 cell related characteristic molecules, such as SOX9Col 鈪，

本文編號：1585078