富血小板血浆对人原代脂肪来源干细胞增殖的影响

Effect of platelet-rich plasma on the proliferation of primary adipose-derived stem cells

目的 观察富血小板血浆对人原代脂肪来源干细胞增殖的影响.方法 脂肪组织及静脉血来源于6例吸脂的女性患者,在无菌条件下,从脂肪组织中分离获取含原代脂肪来源干细胞的血管基质成分;在吸取脂肪的同时,抽取等单位体积的静脉血,离心制备获得富血小板血浆.采用Cellometer细胞计数仪及辅助,吖啶橙-碘化丙啶染色法对血管基质成分活细胞进行细胞计数;血液分析仪对富血小板血浆进行细胞计数.将细胞分为实验组与对照组,两组分别用含5%、10%、15%的富血小板血浆及含10%胎牛血清培养液干预.细胞计数仪检测在24,48,72,96h时,原代脂肪来源干细胞的数量值.结果 ①本实验血管基质成分成骨、成脂转化实验为阳性;Mark检测,第3代脂肪来源干细胞CD73、CD90为阳性,CD45为阴性,逆推验证,可从血管基质成分中,可获取原代脂肪来源干细胞;②通过血清离心法可获得约平均全血4倍的血小板;③实验组较对照组在24,48,72,96h时,ADSCs数量增多,密度增大,其中72,96h时段中,不同浓度的富血小板血浆对脂肪来源干细胞增殖有明显的促进作用,差异具有统计学意义（P ＜0.05）.结论富血小板血浆对体外培养的人原代脂肪来源干细胞的增殖有促进作用.

Objective To observe the effects of platelet-rich plasma on the number, growth rate and adipo- genic differentiation potential of adipose-derived stem cells. Methods Adipose tissue and venous blood was collect- ed from 6 liposuction female patients. Adipose tissue was separated under sterile condition to collect the stromal vas- cular fraction cells that contains adipose-derived stem cells. Platelet-rich plasma was isolated from the unit volume of venous blood of the same liposuction patient by the use of centrifugation. Cellometer analyser and AO-PI staining was used to count the amount of stromal vascular fraction hying cells. Hematology anatlyser was used for cell counting on platelet-rich plasma. In vitro experiment, cells were divided as treatment and control groups. The treatment group contained 5%, 10% and 15% platelet-rich plasma while the control group contained 10% fetal bovine serum as the conditions for culture fluid intervention. Cell counting instruments collected the cell count from the control and treat- ment groups at 24 h, 48 h, 72 h and 96 h. Results ① In this experiment, the stromal vascular fraction osteoblastic and adipegenic conversion test were positive; Detected with Mark test, the expression of CD73 and CDg0 at 3rd gen- eration adipose-derived stem cells was positive while the CD45 was negative. This experiment proved adipose-derived stem cells could be collected from stromal vascular fraction. ② Serumcentrifugation could obtain an average 4 times platelet of whole blood. ③ Compared with the control group, platelet-rich plasma in treatmental group had higher adipose-derived stem cells number with a increased density （P〈0.05） at 24 h, 48 h, 72 h and 96 h. The platelet- rich plasma with diferent concentration at 48 h and 96 h had obviously promotive effect on the adipose-derived stem cells which had a statistical significance （P〈0.05）. Conclusion Platelet-rich plasma can contribute to the growth of primary adipose-derived stem cells in vitro.