富血小板血浆联合人重组骨形态形成蛋白-2诱导羊脂肪基质干细胞成骨化趋势的研究

The experimental study on osteogenic potential of goat adipose derived stromal cells induced by PRP with rhBMP-2

目的探讨如何优化羊等大型哺乳类动物的脂肪基质干细胞（ADSCs）的生物活性及适应体外成骨分化，并建立一种羊脂肪基质干细胞体外诱导成骨的改良方法。方法从2013年3月至2014年9月，取安哥拉羊静脉血制备自体源性富血小板血浆（PRP），并取羊腹部脂肪，经系列原代与传代培养，如下诱导羊骨化脂肪基质干细胞．并分为4组：联合诱导组（富血小板血浆加人重组骨形态发生蛋白-2加ADSCs）、成骨因子组（人重组骨形态发生蛋白-2加ADSCs）、常规诱导组（地塞米松加抗坏血酸加甘油磷酸钠加ADSCs）及非诱导组（空白诱导），第1、3、5、7、9、13、17和21天，倒置显微镜观察，茜素红及VonKossa染色，测定增殖、I型胶原及骨钙素（OC）含量。对各组间差异进行单因素方差分析和最小显著差异t检验，SPSS11．0统计软件进行统计学分析。结果与其他组相比，联合诱导组细胞体积明显增大，胞浆中的细胞器发达，细胞生长旺盛、细胞外基质矿化结节丰富，呈集落状层叠分布：矿化结节出现时间及分布规律：联合诱导组〉成骨因子组〉常规组〉非诱导组；ALP表达时效及强度情况：联合诱导组〉成骨因子组〉常规组〉非诱导组；此外，第13天时联合诱导组增殖活力、I型胶原分别为（0．82±0．19）AU、（79．82±1．36）μg／ml，第17天时0C含量为（78．51±4．32）μg／ml]，均高于其它组，差异有统计学意义（P〈0．05）。结论PRP联合rhBMP-2能通过协同机制，发挥强化ADSCs诱导成骨效应，为一种可行的优化成骨诱导方法。

Objective To evaluate the feasibilty of modified osteogenic culturing of goat adipose derived stromal cells（ADSCs）. Methods From March, 2013 to September, 2014, platelet-rich plasma（PRP） was made from goat autogenous vein blood, and abodimoneal fat was aspirated, following aspetic procedure, primary and se- ries passage of ADSCs was established. Osteoinduced ADSCs was carried out according to following group： combi- native osteoinduction group （PRP＋rhBMP-2 ＋ADSCs）, growth factor group （rhBMP-2＋ADSCs）, conventional induc- tive group（dexamathasone＋ascorbic acid ＋ ADSCs） and noninductive group（blank group）. Converted microscope was used to observe cellular pattern, cell activity, osteocalcin and collagen type I level were detected at 1, 3, 5, 7, 9, 13, 17,21 days. Red Alazarin and Von Kossa staining were also assayed at different interval. Results Under observation of converted microscopy, remarkable cell proliferation with abundant ECM was noticed in combi- native osteoinduction group, compared with other groups, level of cell activity, osteocalcin, collagen type I [ （0.82 ± 2.19）AU, （79.82 ±1.36）U/L at, （78.51 ± 4.32）μg/ml at]were significantly higher than other groups （P 〈 0.05）, and remarkable ALP expression and calcifed nodules were also seen. Conclusion PRP can enhance the inductive effect of rhBMP-2, and combinative osteoinduction procedure acts as a satisfactory cuhuring method.