不同代次骨髓间充质干细胞成骨分化潜能的体外研究

目的观察不同代次人骨髓间充质干细胞(MSC)体外成骨分化能力,为优化组织工程种子细胞提供更多参数。方法应用全骨髓直接贴壁法分离培养10例健康志愿者MSC,依照年龄将MSC分为4组,<20岁,20岁～,30～40岁,>40岁组。取P1(passage 1)～P5诱导成骨分化,诱导分化14d予以碱性磷酸酶染色,21d茜素红染色,Image-Pro Plus软件分析细胞染色阳性区域平均吸光度值。结果 <20岁组P1～P5成骨分化能力无显著差异(P>0.05);30～40岁组P3分化能力低于P1、P2(P<0.05);>40岁组P3分化能力下降更明显(P<0.05or P<0.01),P4、P5几乎分化受阻。结论不同代次的MSC分化能力具有异质性,与年龄有关。>40岁组MSC在超过P3代时,细胞倍增时间明显延长,诱导成功率降低,成骨分化潜能明显下降,选择P2代作为种子细胞更有利于细胞治疗的安全及质量控制。

Osteogenic Differentiation Potential of Different Passages from Bone Marrow Mesenchymal Stem Cells in vitro

Objective To observe the osteogenic differentiation potential of human mesenchymal stem cells(MSC)with different passages in vitro and to provide more parameters for better seed cells source applied to tissue engineering.Methods MSC from 10 donors aged from 14 to 55 years old were isolated using holo-marrow direct inoculation methods.The donors were allocated to four groups according to age:<20,20-,30-40,>40 years old.P1-P5 generation was used for osteoinduction.ALP and Alizarin red staining was performed 14 and 21 days after osteoinduction respectively.The mean density of positive region was analyzed with Image-Pro Plus software.Results There was no significant difference in differentiation potentials of P1-P5 in <20 years group.However,in 30-40 years group,the differentiation potential of P3 was lower than that of P1 and P2(P<0.05),and it was significantly lower in >40 years group(P<0.01).In addition,in >40 years group,the differentiation potential of P4 and P5 was almost arrested.Conclusion The osteogenic differentiation potential of different passages was heterogeneous and this heterogeneity was related to age.In elderly donors(>40 years),when the expansion exceeded three generation times,the doubling time of the cells is significantly prolonged.The osteoinduction rate and differentiation potential were decreased too.So,for the quality control of cell therapy,the second passage was more appropriate.