三种亚型软骨组织来源干细胞的分离培养及鉴定

发布时间：2018-01-03 21:13

  本文关键词：三种亚型软骨组织来源干细胞的分离培养及鉴定　出处：《中国修复重建外科杂志》2015年04期 　论文类型：期刊论文

  更多相关文章： 软骨组织工程 软骨干细胞 软骨亚型 纤维连接蛋白

【摘要】：目的分离培养猪不同亚型软骨组织(弹性软骨、透明软骨以及纤维软骨)来源干细胞并鉴定,为软骨组织工程提供理想种子细胞。方法利用纤维连接蛋白黏附法分别从猪耳软骨、关节软骨以及椎间盘软骨中分离培养干细胞,并进行传代。倒置相差显微镜下观察细胞形态变化,流式细胞术鉴定细胞表面抗原表达水平(阳性标志物CD29、CD90及阴性标志物CD34、CD45),单克隆形成实验鉴定软骨干细胞单克隆形成能力。三向诱导分化鉴定软骨来源干细胞的成软骨、成骨及成脂多向分化潜能。RT-PCR检测成骨(Ⅰ型胶原、Ⅹ型胶原)、成软骨[蛋白聚糖(Aggrecan)、II型胶原]、成脂[脂联素(Adiponectin)、脂肪酸合成酶(fatty acid synthase,FAS)]相关基因表达,并以猪BMSCs作为对照。结果通过纤维连接蛋白黏附法分别从耳软骨(弹性软骨)、关节软骨(透明软骨)、椎间盘软骨(纤维软骨)分选出一群细胞,细胞高表达干细胞表面阳性标志物CD29、CD90,几乎不表达干细胞表面阴性标志物CD34、CD45。经过体外2周培养,单个细胞均能形成细胞克隆。三向诱导分化显示软骨来源的干细胞具备成软骨、成骨和成脂分化能力。RT-PCR结果显示,成骨诱导后关节和椎间盘来源软骨干细胞的Ⅰ、Ⅹ型胶原基因相对表达量明显高于BMSCs(P0.05),耳软骨来源干细胞与BMSCs比较差异无统计学意义(P0.05);成软骨诱导后,3种亚型软骨组织来源干细胞Aggrecan、Ⅱ型胶原基因相对表达量均高于BMSCs(P0.05);成脂诱导后,3种来源软骨干细胞Adiponectin及FAS基因相对表达量均低于BMSCs,但比较差异无统计学意义(P0.05)。结论不同亚型的猪软骨组织中均存在软骨干细胞,具有干细胞的典型特征。
[Abstract]:Objective to isolate and identify the stem cells derived from different subtypes of cartilage (elastic cartilage, hyaline cartilage and fibrocartilage) of pigs. Methods Stem cells were isolated from porcine ear cartilage, articular cartilage and intervertebral disc cartilage by fibronectin adhesion method. The changes of cell morphology were observed under inverted phase contrast microscope and the expression level of cell surface antigen was identified by flow cytometry (positive marker CD29 CD90 and negative marker CD34). CD45, monoclonal formation assay was used to identify the ability of forming chondrocytes. Three ways of inducing differentiation were used to identify the chondrogenic cartilage of chondrogenic stem cells. Detection of osteogenesis (type I collagen, type X collagen, cartilage formation) by reverse transcription-polymerase chain reaction (RT-PCR). [Proteoglycan type II collagen. [Adiponectin (Adiponectin), fatty acid synthase (FAS)). Results A group of cells were isolated from ear cartilage (elastic cartilage), articular cartilage (hyaline cartilage) and intervertebral disc cartilage (fibrous cartilage) by fibronectin adhesion method. The cells expressed CD29 + CD90 on the surface of stem cells and almost no CD34 + CD45 on the surface of stem cells. The cells were cultured for 2 weeks in vitro. Three-way differentiation showed that the chondrogenic stem cells had the ability of chondrogenesis, osteogenesis and adipogenic differentiation. RT-PCR results showed that. After osteogenic induction, the relative expression of type 鈪，

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