人类脂肪来源成体干细胞体外定向成软骨的诱导实验

Experimental induction of human adipose derived adult stem cells （hADASc） into chondrocytes in vitro

目的 评价人类脂肪来源成体干细胞（hADASc）体外培养转基因定向成软骨诱导的可行性.方法 hADASc体外培养后行PGL3.转化生长因子β1（TGF-β1）基因转染并鉴定,检测转染细胞Lueiferase活性相对光读数（RLU）,再行反转录聚合酶链式反应（RT-PCR）检测和抗Ⅱ型胶原免疫组织化学染色（实验组：转染的hADASc,同比对照组：加入软骨细胞定向诱导培养基的未转染hADASc;阴性对照组：未转染的hADASc）,自动图像分析系统分析免疫染色图片并计算阳性颗粒（PU）值.结果 人类皮下脂肪体外分离培养出具有干细胞特征的有核组织细胞.hADASc转染后,实验组测得RLU值（9 212.583±315.240）高于阴性对照组（317.000±20.710,P＜0.01）.RT-PCR结果显示转染的hADASc表达TGF-β1;免疫染色结果显示,实验组与同比对照组呈阳性反应,且两组PU值（13.864±2.416,13.637±2.548）均与阴性对照组的PU值差异有统计学意义（6.013±0.827,P＜0.05）.结论 人类皮下脂肪体外分离培养出具有干细胞特征的有核组织细胞.PGL3-TGF-β1基因转染的hADASc可表达TGF-β1.内源性TGF-β1可诱导hADASc分泌Ⅱ型胶原蛋白,与外源性TGF-β1作用相类似.

Objective To investigate the feasibility of inducing human adipose derived adult stem cells （hADASc） into chondrocytes by gene transfection.Methods hADASc were cultured in vitro, and transfected with gene PGL3-TGF-pl, whose Luceferase activity （RUL value） was measured.PT-PCR was used to monitor the expression of tranfected hADASc.Anti collagen II immunohistochemical staining was performed in the experimental group （transfected hADASc） , active control group （untransfected hADASc cultured in chondrocytes induction medium） and negative control group （untransfected hADASc cultured in conventional medium）.The immuno-staining image was analyzed with an automated imaging analysis system and computed for PU values.Results Nucleated tissue cells with features of stem cells were isolated from in vitro culture of human subcutaneous adipose tissue.The RUL value of the experimental group （9 212.583±315.240） was higher than that of the control group（317.000？0.710, P＜0.01）.RT-PCR revealed that the transfected hADASc could express the TGF-β1.Anti collagen Ⅱ immunohistochemical staining was positive in experimental group and active control group, with PU values （13.864±2.416 and 13.637±2.548,respectively） statistically different from that of the negative control group （6.013 ±0.827, P＜0.05）.Conclusions Nucleated tissue cells with features of stem cells can be isolated from culture of human subcutaneous adipose tissue in vitro.The PGL3-TGF-β1-transfected hADASc may express TGF-β1.The endogenous TGF-β1-induced hADASc produces collagen Ⅱ , with similar actions of exogenous TGF-β1.