压力对骨髓间充质干细胞(BMSCs)膜片复合富血小板纤维蛋白(PRF)双膜结构中BMSCs成软骨能力的影响

摘要

ABM; To investigate the effects of pressure on the chondrogenic differentiation of bone marrowrnmesenchymal stem cells ( BMSCs) in the double membrane of BMSCs combined with platelet - rich fibrin (PRF) (BMSCs/PRF). METHODS: BMSCs were obtained from New Zealand rabbits by density gradient centrifugation, identified by flowcytometry and osteogenic and adipogenic induction experiments. Then BMSCs sheet was prepared. PRF membrane was obtained by centrifuging the auricle arterial whole blood and squeezing the PRF clot. BMSCs sheet was then co - cultured with PRF membrane for 2 days. Afterwards, the BMSCs/PRF compound membranes were stimulated by hydrostatic pressure at 0 kPa (control) ,90, 120 and 150 kPa for 1 and 6 h per day for 2, 4 and 6 days, respectively. Gene expression of PCNA, SOX-9, aggrecan and COL- II of the BMSCs in the BMSCs/PRF compound was examined by real-time PCR . RESULTS: The gene expression of PCNA was promoted by the pressure, especiallyrnat 120 kPa/1 h for 4 days. After 2 consecutive days of pressure treatment, aggrecan was statistically up-regulated, while the expression of SOX-9 and COL-II remained unchanged. However, all the three gene expressions were up -regulated after 4 consecutive days of the pressure treatment, particularly in the 120 kPa/lh group. After 6 consecutive days of pressure treatment, only COL-II gene expression under 90 kPa or 120 kPa for 1 h increased. CONCLUSION: Suitable Pressure stimulation may promote the proliferation and chondrogenic differentiation of BMSCs in the composite of BMSCs/PRF.%目的:观察压力对骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)复合富血小板纤维蛋白(Platelet-rich Fibrin,PRF) (BMSCs/PRF)双膜结构中BMSCs的成软骨能力.方法:密度梯度离心法分离培养兔骨髓间充质干细胞,经表面标记分子检测及成骨、成脂能力鉴定后,制备细胞膜片.将取自同一个体的兔耳廓动脉全血通过离心获得PRF,将PRF压制成膜后与BMSCs细胞膜片复合并进行混合培养.培养2d后按不同力值将复合膜随机分为0 kPa(对照)、90、120、150 kPa 4大组；每一大组再分为刺激1 h/d、6 h/d两组,然后置于加载装置中按分组分别施以不同静态压力.分别于加载第2、4、6天终止培养、取材；Real-time PCR法检测PCNA、SOX-9、Aggrecan、COL-ⅡmRNA表达水平.结果:实验所设定的各组压力刺激条件均可对双膜结构中的BMSCs产生显著的促增殖效应,其中以120 kPa/1 h持续4d的压力条件下PCNA基因表达水平最高.2d压力刺激明显上调Aggrecan水平,而对SOX-9与COL-Ⅱ的表达无显著作用；4d压力刺激可同时促进Aggrecan、SOX-9及COL-Ⅱ mRNA的表达,其中以120 kPa/1 h/4 d的压力条件下的成软骨基因表达水平最高；6 d压力刺激下仅在90、120 kPa加压1h时表现出对COL-Ⅱ mRNA的促进作用.结论:适当的压力刺激可明显促进BMSCs/PRF双膜结构中BMSCs的增殖与软骨向分化能力.