自体富血小板血浆对兔骨髓间充质干细胞增殖的影响

Effect of autologous platelet-rich plasma on proliferation of rabbit bone marrow mesenchymai stem cells

目的建立一种骨髓间充质干细胞（BMSCs）培养新方法，以避免因使用异种血清培养带来的伦理学争议、传播疾病和潜在抗原性等问题，同时满足细胞对各类生长因子的需求。方法经兔耳中央动脉取血并制备富血小板血浆（PRP）。从兔髂骨抽取骨髓，富集BMSCs，分为PRP组、胎牛血清（FBS）组和无血清组，分别采用10％PRP、10％FBS与元血清，配比DMEM．F12培养。观察各组细胞形态、检测细胞增殖活性并绘制生长曲线、表型表达和碱性磷酸酶（ALP）活性。结果3组细胞均在接种后24h左右开始贴壁，并由圆形逐步变为梭形、多角形和不规则形等。3组细胞生长曲线均呈“s”形，PRP组与FBS组增殖明显快于无血清组（P〈0．01），PRP组增殖快于FBS组（P〈0．05）。第4代PRP组与FBS组CD44阳性率分别为88．73％和85．66％、CD90为84．32％和87．58％，均呈阳性表达；CD34为12．38％和14．76％、CIM5为10．47％和11．82％，均呈阴性表达。FBS组和无血清组第1～5代ALP活性相似；但PRP组随着传代而逐渐升高，第4、5代ALP活性（6．42±0．89）U／100ml，（7．02±0．92）U／100ml显著高于其第1、2代（4．27±0．74）U／100ml，（4．70±0．76）U／100ml（P〈0．01），以及高于第4、5代FBS组和无血清组（P〈0．01）。结论自体PRP能有效促进BMSCs增殖，细胞形态、增殖活性及表型与FBS组基本一致；而ALP活性逐代升高，提示PRP有诱导BMSCs向成骨细胞分化的潜能。

Objective To establish a new method to culture bone marrow mesenchymal stem cells （BMSCs）, which can avoid ethic controversy, transmitting diseases, causing immunological antigenicity problems and so on, induced by using heterogeneous serum, and meet the demand on various growth factors. Methods Platelet-rich plasma （PRP） was prepared by using Haemospasia from rabbit＇ s ear central artery. Punctured and sampled bone marrow from rabbit＇ s iliac bone, and collected BMSCs, which divided into PRP group, FBS group and serum-free control group, and cultured in 10% autologous PRP, 10% FBS and serum-free respectively, combined with DMEM-F12 medium. To observe cells morphology, detect proliferation and draw growth curves, cells surface antigen and ALP activity of each group. Results BMSCs of 3 groups adhered to wall after inoculating about 24 hours, presented round and changed into fusiform, polygonal, and irregular forms gradually. The growth curves of 3 groups were ＂S＂ shape, PRP and FBS groups proliferated more rapidly than serum-free control group （P 〈 0. 01 ） , and PRP group more than FBS group（ P 〈 0. 05）. CD44 of the 4th passage BMSCs of PRP and FBS group was 88.73% and 85.66%, and CD90 was 84. 32% and 87.58%, both expressed positively; but CD34 was 12. 38% and 14.76% , and CIM5 was 10. 47% and 11.82% , both expressed negatively. The ALP activity among FBS group and serum-free control group from the 1 st to 5th passage was similar, but the activity of PRP group increased as passaging, and which of 4th and 5th （ 6.42 ± 0. 89 ）, （7.02 ± 0.92） U/100 ml was significantly higher than 1st and 2nd （4. 27 ±0. 74）, （4. 70 ±0. 76） U/100 ml （P 〈0. 01 ）, and higher than the 4th and 5th passage cells of PRP and FBS group（ P 〈 0.01 ）. Conclusion Autologous PRP could promote BMSCs proliferating effectively, the cells morphology, proliferation activity and surface antigen cultured in PRP was similar as in FBS; and the ALP activity increased when it was passaged, which indicated the PRP had the potential to induce BMSCs to osteoblast.