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软骨细胞和骨髓间充质干细胞混合培养构建组织工程软骨的实验研究

发布时间:2019-01-12 09:10
【摘要】: 目的: 探讨软骨细胞和骨髓间充质干细胞(BMSCs)混合培养对构建组织工程软骨的影响,并确定两者的最佳比例。 方法: 取一月龄新西兰兔六只,雌雄不限。体外分离培养扩增兔关节软骨细胞和BMSCs。收获第一代末的软骨细胞和BMSCs,按不同比例(软骨细胞/BMSCs:4/1,2/1,1/1,1/2,1/4,单纯软骨细胞)混合培养一代。以4×107/ml的细胞终浓度接种40μl于聚乳酸聚羟基乙酸聚合物支架( PLGA:4mm×4mm×2mm)上静态培养2天,移入周期性压力场下培养(压力:0~200kPa ,频率:0.1 Hz,时间:8h/d)三周。三周后观察各组组织工程软骨的大小形态质地光泽和组织弹性;HE染色法观察组织工程软骨细胞增殖及分布;甲苯胺蓝染色法观察氨基糖胺聚糖(GAG)的分泌及分布状况,并用1,9二甲基亚甲蓝法定量检测GAG的含量;采用Ⅱ型胶原免疫组织化学法观察Ⅱ型胶原的分泌及分布,并用image-proplus6.0图象分析系统对Ⅱ型胶原染色面积行半定量分析;比色法测定组织工程软骨的DNA含量观察组织工程软骨的细胞增殖情况。 结果: 软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比,体积较大,表面光滑,有弹性,有光泽。组织学显示混合培养组结构致密,细胞外基质分布更均匀,其中2/1组可见软骨陷窝。混合培养组的Ⅱ型胶原染色面积、GAG含量、DNA含量高于单纯软骨细胞组,其中2/1组含量最高,差异有统计学意义(P0.05)。 结论: 软骨细胞和BMSCs混合培养能显著提高组织工程软骨的质量,其中软骨细胞和BMSCs的浓度比为2/1时效果最好。
[Abstract]:Aim: to investigate the effect of co-culture of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) on the construction of tissue engineered cartilage and determine the optimal ratio of the two. Methods: six New Zealand rabbits (male and female) of one month old were selected. Isolation and amplification of rabbit articular chondrocytes and BMSCs. in vitro The first generation of chondrocytes and BMSCs, were harvested and cultured in different proportion (chondrocytes / BMSCs:4/1,2/1,1/1,1/2,1/4, pure chondrocytes). The cells were inoculated with 40 渭 l of 4 脳 107/ml cells in PLGA:4mm 脳 4mm 脳 2mm for 2 days, then cultured under cyclic pressure (pressure: 0~200kPa, frequency: 0. 1 Hz, time: 8h/d) for three weeks. The size of tissue engineered cartilage in each group was observed three weeks later. Form? Texture? HE staining was used to observe the proliferation and distribution of tissue engineered chondrocytes, toluidine blue staining was used to observe the secretion and distribution of aminoglycosaminoglycan (GAG), and the content of GAG was detected by 1- (9) dimethylmethylene blue method. The secretion and distribution of type 鈪,

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