论文部分内容阅读
目的观察在不同滚压参数下,滚压载荷生物反应器对BMSCs-琼脂糖复合体中兔BMSCs向软骨细胞诱导分化的作用。方法分离2.5月龄新西兰兔BMSCs,培养至第3代后与低熔点琼脂糖复合成凝胶块,并在自制模具中形成直径4 mm、高4 mm的圆柱形BMSCs-琼脂糖复合体。将样本分别在0.4 Hz、3 h/d基本参数下比较压缩量10%、20%、30%(分别为A1、A2、A3组),以及0.4 Hz、20%压缩量基本参数下比较20 min、3 h、12 h滚压时间(分别为B1、B2、B3组)对BMSCs向软骨细胞分化的作用,以静态培养作为对照组。在培养24 h及7、14、21 d时取各组标本进行死活细胞检测、实时定量PCR检测、糖胺聚糖(glycosaminoglycan,GAG)含量检测及组织学染色观察。结果死活细胞检测:14、21 d时,A1、A2组活细胞比率均显著高于A3组,B1、B2组活细胞比率均显著高于B3组,差异均有统计学意义(P<0.05)。实时定量PCR检测:各实验组7、14、21 dⅡ型胶原及蛋白聚糖(Aggrecan)mRNA表达均高于对照组,差异有统计学意义(P<0.05);14、21 d时,A1、A2组Ⅱ型胶原、Aggrecan mRNA表达均高于A3组,B1、B2组均高于B3组,差异有统计学意义(P<0.05)。以上指标A1、A2组间及B1、B2组间比较,差异均无统计学意义(P>0.05)。GAG含量检测:14、21 d时,A1、A2组GAG含量均显著高于对照组和A3组,B1、B2组GAG含量也显著高于对照组和B3组,差异均有统计学意义(P<0.05);21 d时A1、A2组间差异均有统计学意义(t=2.830,P=0.027)。组织学观察:21 d时,A2组和B2组出现明显蓝染,可见软骨陷窝样结构;而A1、A3组和B1、B3组蓝染效果较差,分布稀少。结论滚压载荷生物反应器在0.4 Hz、20%压缩量、3 h滚压时间参数下,能更有效地促进BMSCs-琼脂糖复合体中兔BMSCs向软骨细胞诱导分化。
Objective To observe the effect of roller-loaded bioreactor on the differentiation of rabbit BMSCs to chondrocytes in BMSCs-agarose complex under different rolling parameters. Methods New Zealand rabbit BMSCs were isolated from 2.5 months of age. After passage 3, they were mixed with low melting point agarose gel and formed cylindrical BMSCs-agarose complex with diameter 4 mm and height 4 mm in homemade mold. Comparing the parameters of 10%, 20% and 30% (A1, A2 and A3 respectively) under the basic parameters of 0.4 Hz and 3 h / d and comparing the basic parameters of 0.4 Hz and 20% , 3 h, 12 h rolling time (B1, B2, B3 group respectively) on the differentiation of BMSCs into chondrocytes. The static culture was used as the control group. The cells were harvested at 24 h, 7,14 and 21 d respectively for detection of live and dead cells, real-time quantitative PCR, glycosaminoglycan (GAG) and histological staining. Results The viable cells were detected at 14 and 21 d, the ratios of viable cells in A1 and A2 groups were significantly higher than that in A3 group, and the ratios of viable cells in B1 and B2 groups were significantly higher than those in B3 group (P <0.05) . Real-time quantitative PCR showed that the mRNA expression of typeⅡcollagen and aggrecan in experimental group was higher than that in control group at 7, 14, and 21 d (P <0.05), and at 14 and 21 d, A1, The expressions of type Ⅱ collagen and Aggrecan mRNA in group A2 were higher than those in group A3, and those in group B1 and B2 were significantly higher than those in group B3 (P <0.05). There was no significant difference between the above indexes A1, A2 and B1, B2 (P> 0.05). The GAG content of A1 and A2 groups was significantly higher than that of control group and A3 group at 14 and 21 days, and GAG content of B1 and B2 group was also significantly higher than that of control group and B3 group (P <0.05). There was a significant difference between A1 and A2 groups on day 21 (t = 2.830, P = 0.027). Histological observation: At 21 days, there were obvious blue staining in A2 and B2 groups, showing cartilaginous lacunar structures. However, the blue stainings in A1, A3 and B1, B3 groups were poor with less distribution. Conclusion The roller-loaded bioreactor can promote the differentiation of rabbit BMSCs into chondrocytes in BMSCs-agarose complex under 0.4 Hz, 20% compression and 3 h rolling time.