微重力条件下动态三维诱导骨髓间充质干细胞分化为软骨细胞:与静态培养的比较

Dynamic three-dimensional induction and differentiation of marrow mesenchymal stem cells into chondrocytes under microgravity:Comparison with static culture

目的:观察微重力条件下动态三维诱导骨髓间充质干细胞向软骨细胞的分化,并与静态培养作比较。方法:实验于2005-08/2006-04在哈尔滨医科大学附属第一医院中心实验室完成。穿刺抽取2月龄新西兰大白兔骨髓,密度梯度离心法分离纯化,体外培养扩增。取第3代骨髓间充质干细胞分组诱导培养:三维动态培养组:1%藻酸钠溶液洗涤并重新悬浮细胞,细胞密度为5×1010L-1,滴入200mmol/L氯化钙溶液,立即形成藻酸钙凝胶微球,细胞被悬浮固定于球内部,静止5min,取出凝胶微球,磷酸盐缓冲液充分洗涤,置于旋转式细胞培养系统,微重力条件下动态诱导。二维动态培养组:细胞直接接种于平面培养瓶,置于旋转式细胞培养系统,微重力条件下动态诱导。三维静态培养组:藻酸钙凝胶微球悬浮细胞静态培养。二维静态培养组:细胞直接接种于平面培养瓶静态培养。诱导培养2周后取材,甲苯胺蓝染色、Ⅰ、Ⅱ型胶原免疫组织化学染色,测定胶原和蛋白多糖含量。结果:①藻酸钠接触钙离子迅速形成透明凝胶微球,直径约为1.0mm,均匀一致,有光泽,易于操作。倒置显微镜下见凝胶微球中的细胞呈球形,核仁清晰,动、静态培养后立体结构相似。甲苯胺蓝染色阳性,表达Ⅱ型胶原,无明显Ⅰ型胶原表达。平面培养组细胞形态由梭形向多角形、多边形转变,核周可见黑色颗粒。甲苯胺蓝染色弱阳性,仅少数细胞表达Ⅱ型胶原。②与静态培养相比,动态培养出现明显软骨分化,动态培养组胶原和蛋白多糖产量均高于静态培养培养组,以三维动态培养组效果最佳[胶原:0.078±0.004,0.069±0.003,0.048±0.002,0.035±0.004;蛋白多糖:0.111±0.003,0.092±0.002,0.069±0.003,0.058±0.002,(P<0.05)]。结论:立体诱导优于平面诱导,微重力动态培养可提高细胞诱导分化质量。

AIM： To observe the induction and differentiation of marrow mesenchymal stem cells （MSCs） into chondrocytes under dynamic microgravity and three d.imensions （3D）, and compare it with static culture. METHODS： The experiment was conducted in the Central Laboratory of the First Affiliated Hospital of Harbin Medical University from August 2005 to April 2006. The blood of bone marrow was taken from the two-month New Zealand rabbits under anesthesia, then the MSCs were isolated from bone marrow, purified by density gradient centrifugation, and then cultured and amplified in vitro. The MSCs of the 3^rd passage were induced into chondrocytes at four different groups： 3D dynamic culture group： The cells were washed with 1% algin solution and renewed to suspend cells with density of 5×10^10 L^-1; after 200 mmol/L calcium chloride solution was dropped in, the calcium alginate gelatum microballoons immediately formed, and the cells were floated and fixed in microballoons. Five minutes later, the mingelatum microballoons were dislodged and put in the＇ revolve cell culture system （RCCS） after washed with PBS for dynamic culture under microgravity. 2D dynamic culture group： The cells were inoculated directly in a culture flask in RCCS for a dynamic culture under microgravity. 3D static culture group： The cells in the calcium alginate gelatum ware inoculated directly in a culture flask for static culture. 2D static culture group： The cells ware inoculated directly in a plane culture flask for static culture. Each group was cultured for 2 weeks and prepared slices for toluidine blue dyeing, and collagen Ⅰ, Ⅱ immunohistochemical staining to measure the content of collagen and proteoglycan products. RESULTS： ①Algin contacted Ca^2＋ formed transparent gelatum microballoons quickly of lustered, 1.0 mm in diameter. The cells were globular with clear nucleoles, and the stereochemical structure after dynamic and static culture was similar with that of normal chondrocytes. Toluidine blue staining was positive with collagen Ⅱ but no collagen Ⅰ. Cell morphous of 2D culture was transformed from fusiform to polygon with black particles around the nucelus. Toluidine blue dyeing was weakly positive, and only a little cells expressed collagen Ⅱ. ②Compared with static culture, cartilage differentiation was found in the dynamic culture groups, and the content of collagen and proteoglycan products in the dynamic culture groups ware higher, especially the 3D dynamic culture group [collagen： （0.078±0.004, 0.069±0.003, 0.048±0.002, 0.035±0.004）A; （0.111±0.003, 0.092±0.002, 0.069±0.003, 0.058±0.002）A, P 〈 0.05]. CONCLUSION： 3D culture induction is superior to 2D ones, and the dynamic culture under microgravity could improve the differentiation quality of cells.