骨髓间充质干细胞体外成软骨分化的形态学变化

Morphological characteristics of bone marrow mesenchymal stem cells differentiating into chondrocytes in vitro

背景:骨髓间充质干细胞在向软骨细胞分化过程中可形成聚合体,但是该形态学特点是否可代表骨髓间充质干细胞已经成软骨分化尚无研究报道。目的:观察骨髓间充质干细胞成软骨分化过程中聚合体的形成和成软骨分化情况。方法:体外培养大鼠骨髓间充质干细胞,传代至第6代时备用。对照组用无血清LG-DMEM培养基培养14 d,诱导组用含转化生长因子β3的成软骨完全诱导培养基培养14 d,倒置显微镜观察诱导过程中细胞形态变化;免疫荧光染色、甲苯胺蓝染色、qR T-PCR检测成软骨特异指标Ⅱ型胶原、蛋白多糖和成软骨转录因子SOX9的基因表达及蛋白合成情况。结果与结论:(1)与对照组相比,诱导组骨髓间充质干细胞在第3天失去典型的成纤维形态,第7天开始形成成簇的单层聚合体,第14天形成直径更大的多层聚合体;(2)聚合体的免疫荧光染色及甲苯胺蓝染色均阳性,而聚合体外的单个细胞无特异染色;(3)qR T-PCR亦证实诱导组有成软骨特异基因Ⅱ型胶原、蛋白多糖和SOX9表达;(4)上述研究结果提示骨髓间充质干细胞成软骨分化过程中聚合体的形成可作为成软骨分化的一种形态学检测指标。

BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)can compact to form aggregates during chondrogenic differentiation,but whether the formation of aggregation is a prerequisite for chondrogenic induction is not yet documented.OBJECTIVE:To observe the morphological characteristics of BMSCs differentiating into chondrocytes.METHODS:BMSCs were cultured in vitro and passage 6 cells were used for the following experiments.Cells were induced in serum-free LG-DMEM medium as control group or in complete chondrogenic induction medium containing transforming growth factorβ3 as induction group for 14 days.Cellular morphology during differentiation process was observed by inverted microscope.Chondrocyte-specific markers,including type II collagen,aggrecan and chondrogenic transcription factor SOX9,were evaluated by immunofluorescence,toluidine blue staining and quantitative RT-PCR.RESULTS AND CONCLUSION:Compared with the control group,the cells began to lose the typical morphology at day 3 of chondrogenic induction,and then compacted to form mono-layered aggregates and multi-layered aggregates at days 7 and 14,respectively.The aggregates were positive for immunofluorescence and toluidine blue staining,while no staining was detected in the single cells.qRT-PCR results also confirmed the mRNA expression of type II collagen,aggrecan and SOX9 in the cells after chondrogenic induction.Therefore,these abovementioned results suggest that the formation of aggregation in BMSCs can be one of morphological markers of chondrogenic differentiation.