中文摘要：

目的：探索人间充质干细胞（human mesenchymal stem cells,hMSCs）成软骨细胞诱导分化能力评价技术和评价策略,为hMSCs生物学有效性体外评价提供方法学参考。方法：高密度体外接种hMSCs,促使其形成软骨样3D微球结构,并在此基础上对其成软骨细胞诱导分化能力进行评价。评价内容有：（1）微球的自发形成能力以及成软骨诱导分化过程中外观形态学分析。（2）以阿辛蓝及番红O染色为基础的组织结构分析,用于软骨细胞外基质形成能力分析。（3）通过Real-time PCR对软骨细胞特定基因（ACAN,COL1A2,COL2A1,COL10A1和MIA）表达水平的分析。在对以上评价结果综合分析和软骨组织学方法验证的基础上,对hMSCs体外成软骨细胞诱导分化能力进行综合准确评价。结果：具备软骨细胞诱导分化能力的hMSCs可表现为：（1）具有自发形成软骨样细胞微球能力,并在诱导分化过程中形成白色光滑致密软骨样结构,阿辛蓝染色呈深蓝色。（2）组织学上可观察到细胞微球外周呈同心圆排列的软骨细胞外基质层。（3）成软骨诱导分化过程中软骨细胞特定基因表达显著上调。不同hMSCs在上述三方面的综合特性上存在较大差异。结论：对hMSCs成软骨细胞诱导分化过程中的形态学、组织学和特定基因表达水平的检测较单一方法的检测能够更加客观准确地评价hMSCs的成软骨细胞分化能力。

英文摘要：

Objective： To explore a comprehensive assessment methodology and strategy for evaluating chondrogenic differentiation of human mesenchymal stem cells（ hMSCs）,thus to provide technical support for evaluation of the in vitro biological effectiveness of hMSCs. Methods： Chondrocyte-born microballs were formed by culturing hMSCs in high density in form of droplets in cell culture dish. The microballs were then put in the in vitro environment of chondrogenic differentiation and analyzed by three approaches,i. e.,further forming chondrocyte tissues with typical morphological characteristics,staining intracellular matrix derived from chondrocyte tissues by Alcian blue and Safranin O,and measuring the changes in expression of chondrocyte-specific genes,i. e. ACAN,COL1A2,COL2A1,COL10A1,and MIA,by real-time PCR. At the end,after validating by histological analysis,the capability of chondrogenic differentiation of hMSCs was evaluated by all the three approaches together. Results：The hMSCs with potential of chondrogenic differentiation exhibited the following features： formation of microballs spontaneously after the cells being congregated in high density and further condensation of the microballs with smooth appearance and positive Alcian blue staining in the condition of chondrogenic differentiation; formation of typical chondrocyte intracellular matrix showing positive staining by Alcian blue and arrangement in concentric circles at the periphery of the chondrocyte microballs; dramatic up-regulation of chondrocyte specific genes following the treatment of chondrogenic differentiation. Moreover,hMSCs of different donors and tissue origins exhibited significant difference in each of all three aspects chondrogenic differentiation. Conclusion： The evaluation technology employing three assessment approaches in combination,which covers the morphology,histology and gene expression associated with chondrocyte characteristics,is much more objective and accurate than single approach in evaluating the potential of chond