兔脂肪来源干细胞分离培养及富血小板血浆对其增殖的影响

The isolation and cultivation of the rabbit adipose-derived stem cells and the effects of platelet-rich plasma on the proliferation of those cells

目的探讨自体富血小板血浆（PRP）对脂肪来源干细胞（ADSCs）增殖的影响。方法选取新西兰大白兔的颈背部区脂肪垫，采用贴壁法及I型胶原酶消化法行体外培养兔脂肪来源干细胞，观察其形态特征；取P3代细胞进行成脂、成骨诱导分化，以油红O、茜素红染色鉴定；抽取兔心脏血，经离心处理后制备富血小板血浆，行血小板计数；采用CCK-8法检测不同作用时间（24、48、72h）对脂肪来源干细胞增殖活动的影响。结果兔脂肪来源干细胞原代及传代细胞呈长梭或多边形贴壁生长；油红O及茜素红染色呈阳性；全血血小板计数为（313．0±137．5）×10^9／L，PRP血小板计数为（1267．0±760．2）×10^9／L，PRP血小板计数是全血的4．08倍；CCK-8法显示PRP组在24、48、72h较对照组增殖明显（P〈0．01）。结论PRP对体外培养的兔脂肪来源干细胞的增殖有明显的促进作用。

Objective To investigate the effects of the autologous platelet-rich plasma （PRP） on the proliferation of the adipose-derived stem cells （ ADSCs ）. Methods The cervieodorsal adipose tissue of the New Zealand white rabbit was resected. The ADSCs were cultured in vitro with adherence method and type I collagenase method. The morphological characteristics of the ADSCs were observed. The passage 3 ADSCs were inducted into adipogenesis and osteogenesis, and they were detected with oil red 0 and alizarin red staining. The rabbit heart blood was extracted and prepared by centrifugation for making PRP, and the platelets were counted by platelet cell count board. The effects of the three groups on the proliferative activities of the ADSCs were detected by the CCK-8 （ cell counting kit-8 ） method at different times （24, 48, 72 hours）. Results The rabbit ADSCs in primary and passage ceils grew in the way of long spindle or polygonal adherence. The results of oil red 0 and alizarin red staining of the passage 3 ADSCs were positive. The number of the platelet in the whole blood was （313.0 ± 137.5 ） ×10^9/L, while in the PRP was （ 1267.0 ± 760.2） ×10^9/L, which was 4.08 times of that in the whole blood. CCK-8 method showed that the proliferative activity of the PRP group was obviously stronger than that in the control group at 24, 48, 72 hours （ P 〈 0.01 ）. Conclusion The proliferation of rabbit ADSCs could be enhanced by autologous PRP in vitro.