摘要
目的研究体外培养rBMSCs经TGF-β1诱导分化的软骨细胞复合左旋聚乳酸\β-磷酸三钙(PLLA\β-TCP)多孔支架材料体外构建仿生人工软骨。方法低温挤出成形法制备成PLLA\β-TCP复合多孔支架材料,体外分离、培养rBMSCs至第3代,利用含有TGF-β1特殊诱导系统诱导其向软骨细胞分化,诱导14d后用甲苯胺蓝染色及II型胶原免疫组化进行鉴定后与PLLA\β-TCP多孔支架材料体外复合培养,并取第7、14、21d细胞复合材料进行电镜扫描观察细胞贴附、生长、增殖状况,同时消化收集贴附支架第7、14、21d的细胞,行RT-PCR检测分化软骨细胞相关基因aggrecan、Col2A1在mRNA水平的表达,Western-bolt检测II型胶原蛋白的分泌情况。结果 rBMSCs经诱导后向软骨细胞分化,甲苯胺蓝染色见分化软骨细胞分泌糖胺聚糖(glycosaminoglycan,GAG),Ⅱ型胶原免疫组织化学染色呈阳性;电镜扫描见分化细胞在支架材料分布均匀,黏附良好;RT-PCR及Western-bolt检测示7、14、21daggrecan、Col2A1在mRNA水平、II型胶原蛋白均有不同程度表达。结论利用含有TGF-β1特殊诱导系统诱导rBMSCs分化的软骨细胞复合到PLLA\β-TCP多孔支架材料上,细胞生长良好,并能正常分泌软骨细胞特异细胞外基质,体外成功构建了组织工程软骨。
Objective To investigate the effects of PLLA／β-TCP scaffold guided chondrocyte differentiation of rabbit BMSCs induced by TGF-β1 in vitro. Methods Low-temperature extrusion skill was taken to prepare PLLA／β-TCP porous scaffolds. rBMSCs were isolated and cultured, followed by TGF-β1 induced differentiation into chondrocytes about 14 days. Toluidine blue and type II collagen immunohistochemistry staining were use to identify induced cells. And then, cells were seeded into PLLA／β-TCP scaffolds. scanning electron microscopy（SEM） was used to observe attached cells at the culturing time of 7, 14, 21 days. RT-PCR was used to detect mRNA expression of aggrecan and Col2A1,as well, western blot was used to detect expression of type II collagen of seeded cells. Results rBMSCs were successfully induced into chondrocytes under the stimulation of TGF-β1. Toluidine blue staining demonstrated glycosaminoglycan（GAG） in induced cells. type II collagen positive cells was popular. SEM observation confirmed the scaffold was favored for cell adhesion. Aggrecan, Col2A1 and type II collagen expressed in induced and scaffold seeded cells. Conclusions TGF-β1 is feasible to induced rBMSCs into chondrocytes, furthermore, induced cells is compatible with PLLA／β-TCP porous scaffolds.
出处
《中国临床解剖学杂志》
CSCD
北大核心
2012年第5期543-547,共5页
Chinese Journal of Clinical Anatomy
基金
深圳市卫生局科技计划重点项目资助(201001013)
深港创新圈资助计划项目(SG200810200101A)
