软骨细胞上清液诱导骨髓间充质干细胞分化的软骨细胞的形态学及功能检测

摘 要］目的：应用家兔肋软骨细胞上清液诱导骨髓间充质干细胞（MSCs）向软骨细胞定向分化，并对分化的细胞进行形态学和功能检测，探讨MSCs在体外成软骨分化的条件。方法：分离家兔的MSCs和肋软骨细胞进行体外培养，收集前3代肋软骨细胞上清液作为诱导液，对第3代MSCs进行14 d的诱导培养。设置实验组、阳性对照组和阴性对照组，观察各组细胞的形态，甲苯胺蓝染色观察特异性蛋白表达、各组ALP活性，RT-PCR法检测各组细胞中的Ⅱ型胶原(ColⅡ) mRNA的表达。结果：经过14 d的诱导培养，实验组MSCs由长梭形逐渐变成多角形，甲苯胺蓝染色呈阳性；ALP活性显著升高，与其他2组比较差异有统计学意义(P＜0.01）；ColⅡ mRNA表达呈阳性。阴性对照组甲苯胺蓝染色呈阴性，ColⅡ mRNA表达呈阴性。阳性对照组甲苯胺蓝染色呈阳性，ColⅡ mRNA表达呈阳性。结论：家兔肋软骨细胞上清液能够促进MSCs向软骨细胞方向分化。

Morphology and function detection of chondrocytes differentiated from bone marrow mesenchymal stem cells induced by supernatant of chondrocytes

Objective To induce bone mesenchymal stem cells(MSCs) of rabbits to differentiate directionally into chondrocytes in vitro and to observe the morphology of cells and detect the function,and to discuss the necessary differentiation condition in vitro.Methods The MSCs and chondrocytes were isolated from rabbits and cultured in vitro.The supernatant of chondrocytes in the first three passages were collected and used to induce transformation of MSCs for 14 d.Three experimental groups were set up: negative control group,experimental group and positive control group.After 14 d of induction,the morphology of the cells in each group was observed,and the specific proteins were observed by toluidine blue staining,and the alkaline phosphatase(ALP) activities were detected,and the collegen Ⅱ(Col Ⅱ) mRNA expressions were detected by RT-PCR.Results 14 d after induction,compared with negative control group,the induced MSCs in experimental group changed into polygonal shape from spindle shape and there was positive appearance of toluidine blue staining,the ALP activity and Col Ⅱ mRNA expression were increased significantly compared with the other two groups(P<0.01).In negative control group,there was negative appearance of toluidine blue staining and Col Ⅱ mRNA expression.In positive control group,there was positive appearance of toluidine blue staining and Col Ⅱ mRNA expression.Conclusion The supernatant of chondrocytes from rabbits can promote the MSCs differentiate into chondrocytes in vitro.