GDF-5在软骨细胞再分化中的作用及机制研究

发布时间：2018-03-08 18:36

  本文选题：关节软骨细胞　切入点：生长分化因子5　出处：《新疆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：观察体外传代过程中C57小鼠关节软骨细胞去分化现象,GDF5作用于已去分化的软骨细胞,使其再分化。兔软骨细胞三维培养,GDF5诱导后修复兔膝关节缺损,行检测进一步证明GDF5的作用。初步研究MiRNA-145在软骨再分化中的作用机制。方法：新生C57小鼠膝关节分离获取原代软骨细胞传代至P7代,对P1、P4代和GDF5诱导的P4代细胞从形态、增殖、基质分泌以及基因和蛋白表达等方面采用显微镜观察,CCK-8增殖曲线测定、细胞阿利辛蓝染色、qRT-PCR及Western Blot进行比较。将不同实验组的兔软骨细胞与PLGA/CHI络合物支架结合7天后修复兔膝关节缺损,三月后取材,行HE、阿利辛蓝、C012免疫组化染色等检测。将MiRNA-145的模拟剂和抑制剂转染入细胞,检测胞内SOX9水平。结果：C57小鼠及兔关节软骨细胞传代过程中形态发生变化；P4代细胞增殖能力较P1代稍减弱,P7代无明显增殖；P1代胞外基质分泌量高于P4代,而P4代经GDF5诱导7天后表达量有所上升；P4代细胞相关特性分子(SOX9、Col Ⅱ和Aggrecan等)mRNA及蛋白表达水平较P1代下调,经GDF5诱导后表达水平回升；PLGA/CHI支架的细胞毒性低且细胞可均匀分布材料各层。P4细胞/材料GDF5诱导组修复缺损效果介于P1细胞/材料组和P4细胞/材料组之间；抑制MiRNA-145可增强SOX9的表达,反之亦然。结论：体外培养的P4代软骨细胞出现较明显的去分化现象,经一定浓度(300ng/ml)的GDF5单层诱导7天,SOX9、Col Ⅱ及Aggrecan含量回升,体内细胞+PLGA/CHI支架复合物修复软骨缺损的各项检测结论一致。MiRNA-145对软骨细胞去分化过程中SOX9的表达具有负调控作用。
[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，

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