Choukroun’SPRF对体外培养人脂肪干细胞增殖及成骨分化的影响

目的：观察自体富血小板纤维蛋白Choukroun＇s PRF（Choukroun＇s platelct-rich fibrin）对体外培养人脂肪来源干细胞（adipose-derived stem cells,ADSCs）增殖及成骨分化能力的影响。方法：取吸脂术者自愿捐献的脂肪组织分离培养ADSCs，采用Choukroun法制备自体PRF备用，观察细胞生长情况。取第3代ADSCs分别向骨细胞、脂肪细胞、神经球细胞定向诱导分化鉴定，并行细胞表面抗原CD29、CD45、CD90流式检测鉴定。将第3代ADSCs分别采用含PRF的普通培养基（PRF组Ⅰ）和不含PRF的普通培养基（对照组Ⅰ）进行培养，观察细胞生长情况，培养1天、3天、5天、7天后采用CCK-8试剂检测细胞增殖活性。另外分别采用含PRF的成骨诱导培养基（PRF组Ⅱ）、不含PRF的成骨诱导培养基（对照组Ⅱ）及不合PRF的普通培养基（空白组）进行培养，第7天、14天、21天、28天行碱性磷酸酶活性（ALP活性）检测；诱导细胞培养后第7天、14天天各组分别行von Kossa染色观察钙结节形成情况。结果：第3代ADSCs倒置显微镜下观察大多呈梭形，向骨细胞、脂肪细胞、神经干细胞定向诱导鉴定均为阳性，流式检测鉴定CD29、CD90为阳性，CD45为阴性。CCK-8法示PRF组Ⅰ的0D值均大于对照组Ⅰ，两组比较差异均有统计学意义（P〈0．01）。ALP活性检测示PRF组Ⅱ第7天、14天、21天、28天细胞活性较对照组Ⅱ均大，两组比较差异均有统计学意义（P〈0．01）。PRF组Ⅱ成骨诱导7天后vonKossa染色阳性；14天后阳性细胞增多，对照组Ⅱ诱导7天未见钙结节，14天见少量阳性钙结节，空白组培养14天未见黑色钙结节。结论：Choukroun’sPRF明显促进脂肪干细胞增殖及成骨分化，为骨组织工程提供了新的技术。PRF与干细胞共同培养可能还有许多潜在的临床及生物工程应用价值，值得进一步研究。

The effect of Choukroun's PRF on the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro

Objective To observe the effect of Choukroun＇s PRF （Choukroun＇s platelet-rich fibrin） on the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro. Methods Human ADSCs （adipose- derived stem cells） were isolated from adipose tissue obtained from donor undergoing liposuction and were cultured, and autologous PRF was prepared by Choukroun method,and growth condition of ADSCs was observed.ADSCs at passage 3 were cultured in adipogenic,osteogenetic and neurospheres differentiation medium and underwent identification,flow cytometric analysis for cell surface antigen CD29,CD45 and CD90 were performed. ADSCs at passage 3 cultured by common culture medium containing PRF （PRF group Ⅰ ）, and cultured by common culture medium without PRF （control group Ⅰ）. Growth condition of the cells was observed by inverted microscope. CCK-8 method was used to observe cell proliferation activity 1,3,5,7 days after culture. Then ADSCs cultured by osteogenic induction culture medium containing PRF （PRF group Ⅱ）, cultured by osteogenic induction culture medium without PRF （control group Ⅱ ） and cultured by common culture medium without PRF （blank group）.ALP activity detection was conducted 7,14,21 and 28 days after culture. Von Kossa staining was performed on the three groups 7 and 14 days after culture to detect the formation of calcium nodule. Results Most ADSCs at passage 3 were spindle-shaped under the inverted microscope. Adipogenic , osteogenetic and neurospheres differentiation were positive, and flow cytometric analysis of CD29 and CD90 were positive,CD45 were negative. CCK-8 method revealed the Optical Density value of PRF group Ⅰ were all greater than control group Ⅰ ,and there were significant diferences between two groups （P〈0.01）. ALP activity detection demonstrated the cell activity value of PRF group Ⅱ at 7, 14, 21 and 28 days was were all greater than control group II, and there were significant diferences between two groups （P〈0.01）. At 7 days after osteogenic induction,Von Kossastaining showed the formation of black calcium nodule in PRF group Ⅱ ,and after 14 days,the black calcium nodule weremore. Control group Ⅱ were not observed the black calcium nodule,and were observed a little. The blank group were still not observed after 14 days. Conclusion Choukroun＇s PRF can significantly improve the proliferation of human ADSCs and induce their osteogenic diferentiation in vitro, providing a new source of technology for bone tissue engineering. The potential clinical and Biological Engineering applications of a PRF a.nd ADSCs conglomerate are numerous,and it was worthed for further research.