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目的:观察自体富血小板纤维蛋白Choukroun’s PRF(Choukroun’s pl at el et-ri ch fi bri n)对体外培养人脂肪来源干细胞(adi pose-deri ved st em cel l s,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)及不含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OD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von Kossa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’s PRF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。
OBJECTIVE: To investigate the effect of Choukroun’s pl at el et-ri ch fi bri n on the proliferation and osteogenic differentiation of adi pose-derived vestigial stem cells (ADSCs) in vitro influences. Methods: ADSCs were isolated and cultured from adipose tissue donated by liposuction patients. The autologous PRF was prepared by Choukroun method and the cell growth was observed. The third generation ADSCs were induced to differentiate into osteoblasts, adipocytes and neurospheres respectively. Flow cytometry was used to detect the expression of CD29, CD45 and CD90. The 3rd generation of ADSCs were cultured with ordinary culture medium containing PRF (PRF group Ⅰ) and ordinary culture medium without PRF (control group Ⅰ), respectively. The growth of cells were observed and cultured for 1 day, 3 days, 5 days, 7 Day after the use of CCK-8 reagent to detect cell proliferation activity. In addition, osteoblast-inducing medium (PRF group II), PRF-free osteoblast-inducing medium (control group II) and PRF-free ordinary medium (blank group) were cultured separately on the 7th day, The alkaline phosphatase activity (ALP activity) was detected on day 14, day 21, and day 28; von Kossa staining was performed on the 7th day and the 14th day after induction of cell culture to observe the formation of calcium nodules. Results: The third generation of ADSCs observed under inverted microscope mostly fusiform, osteoblasts, adipocytes, neural stem cells orientation-induced identification were positive, flow cytometry identification of CD29, CD90 positive, CD45 negative. The CCK-8 method showed that the OD value of PRF group Ⅰ was larger than that of control group Ⅰ, the differences between the two groups were statistically significant (P <0.01). ALP activity test showed that the cell viability of PRF group Ⅱ was significantly larger than that of control group Ⅱ at day 7, day 14, day 21, and day 28 (P <0.01). After 7 days of osteogenic induction, von Kossa staining was positive in PRF group Ⅱ. Positive cells increased after 14 days, while in control group Ⅱ, no calcium nodules were found in 7 days and a small amount of positive calcium nodules in 14 days. Festival. Conclusion: Choukroun’s PRF can significantly promote the proliferation and osteogenic differentiation of adipose-derived stem cells, which provides a new technique for bone tissue engineering. PRF and stem cells co-culture may have many potential clinical and bio-engineering value, it is worth further study.