重组人骨形成蛋白2诱导脂肪成体干细胞向软骨分化的形态学研究

目的 探索分离培养兔脂肪成体干细胞，经重组人骨形成蛋白2（recombinant human bone morphogenetic protein2，rhBMP-2）定向诱导后，向软骨细胞分化的可能。方法 将4月龄新西兰兔脂肪组织机械分割，通过Ⅰ型胶原酶消化后得到脂肪成体干细胞（adipose-derived adult stem cells，ADASCs），采用微球方法进行培养，在rhBMP-2诱导组、重组人转化生长因子β1（recombinant human transforming growth factor β1，rhTGF-β1）诱导组和rhBMP-2＋rhTGF-β1（共同诱导组）干预6d后，各组诱导液中分别加入维生素C50μg／ml，再干预8d，应用HE、Alcian blue、Von kossa染色和Ⅱ型胶原免疫组织化学染色等组织学方法对诱导后的细胞进行鉴定。结果 连续诱导培养14d后，各诱导组HE染色显示软骨陷窝形成，对照组软骨陷窝形成不明显。Alcian blue染色见细胞基质中富含蛋白多糖，胞外基质分泌较多的Ⅰ型胶原；但Von kossa染色为阴性；对照组均为阴性。结论 在微球方法培养下，通过相关形态学研究表明，rhBMP-2具有诱导脂肪成体干细胞向软骨细胞分化的能力，将为采用ADASCs修复软骨损伤奠定理论基础。

A MORPHOLOGICAL STUDY OF CHONDROGENESIS BY ADIPOSE-DERIVED ADULT STEM CELLS INDUCED BY RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2

OBJECTIVE: To investigate the possibility of differentiation of the isolated and cultured adipose derived adult stem cells into chondrocytes, which is induced by the recombinant human bone morphogenetic protein 2 (rhBMP 2). METHODS: The rabbit adipose tissue was minced and digested by collagenase Type I. The adpose derived adult stem cells were obtained and then they were cultured in the micropellet condition respectively in the rhBMP-2 group, the rhTGF-beta1 group, the combination group, and the control group for 14 days. The differentiation of the adipose-derived stem cells into chondrocytes was identified by the histological methods including HE, Alcian blue, Von kossa, and immunohistochemical stainings. RESULTS: After the continuous induction by rhBMP-2 and continuous culture for 14 days, the HE staining revealed a formation of the cartilage lacuna; Alcian blue indicated that proteoglycan existed in the extracellular matrix; the immunohistochemical staining indicated that collagen II was in the cellular matrix; and Von kossa indicated that the adipose-derived stem cells could not differentiate into the osteoblasts by an induction of rhBMP-2. CONCLUSION: In the micropellet condition, the adipose-derived adult stem cells can differentiate into the chondrocytes, which is initially induced by rhBMP-2. This differentiation can provide a foundation for the repair of the cartilage injury.