不同方法制备兔自体富血小板血浆凝胶支架促进脂肪干细胞增殖比较

目的探讨不同浓度及离心方法制备自体富血小板血浆（platelet rich plasma，PRP）凝胶支架对脂肪干细胞（adipose derived stem cells，ADSCsl增殖及生长因子释放的影响。方法取新西兰大白兔动脉血分别采用二次及三次离心法制备自体富血小板血浆支架，检测其中的血小板浓度，同时通过添加DMEM培养基将PRP中血小板绝对浓度调整为相同的1000×109／L，之后继续向其中加入培养基将PRP按体积分数调整为30％、40％、50％、60％的不同浓度，并设置仅含培养基的空白对照组。之后向各组PRP中植入经荧光染色的脂肪干细胞（ADSCs）。7d内每日观察计数细胞数目绘制细胞生长曲线，在第2、3、4、6天用MrITr比色法分析各组脂肪干细胞的存活和增殖能力，通过ELISA定量检测培养7d后PRP脂肪干细胞复合体中生长因子TGF—B1及细胞的Ⅱ胶原含量。结果二次离心法及三次离心法所制备的PRP中血小板浓度分别为（1320．5±227．31×109／L及（1724．5±316．31×10。／L，为全血中血小板浓度的5．4及7．1倍，三次离心法所制备的PRP中血小板浓度明显高于二次离心法（P〈0．05）。采用两种离心方法所制作的PRP细胞复合体中的脂肪干细胞数目均在其PRP其中血小板浓度为500×109／L时达到最高值，分别为（1．02±0．13）×104及（1．00±O．14）×10。（P〉0．05）。各组不同浓度的PRP浓度的PRP细胞复合体中当其中血小板浓度达500×109／L时其中脂肪干细胞数目、活性均为最高，且其中的TGF—B1及细胞的Ⅱ胶原含量也高于其他各组俨〉0．05）。结论三次离心法制备PRP，血小板收集率较二次离心法高，但在促进脂肪干细胞增殖上无明显差异。PRP中血小板浓度达500×109几左右时，其中的脂肪干细胞增殖及活性达到最高，可能是制备PRP脂肪干细胞复合体的最适浓度。

Auto-platelet rich plasma gel stents produced with different methods promote proliferation of adipose-derived stem cells

Objective To study the effect of auto- platelet rich plasma （PRP） gel stents produced by centrifugation on proliferation of adipose derived stem ceils （ADSCs） and release of growth factors. Methods Auto-PRP gel stents were produced by two or three centrifugations of arterial blood from New Zealand rabbits. Platelet concentration in such stents were measured. After the absolute platelet concentration in auto PRP gel stents was adjusted to 1 000 x 109/L by adding DMEM, the volume fraction of auto-PRP gel stents into which DMEM was added was adjusted to 30%, 40%, 50% and 60% of the absolute platelet concentration. Auto-PRP gel stents containing only DMEM served as a blank control group. Fluorescence- stained ADSCs were then implanted into auto-PRP gel stents and counted for 7 consecutive days. Cell growth curve was plotted. Survival rate and proliferation activity of ADSCs were analyzed by MTT colorimetry on days 2-4 and 6. TGF- [3 1 and collagen- ff levels in ADSCs were measured by ELISA. Results The platelet concentration in auto-PRP gel stems produced by two and three centfifugations was（1 320.5 ± 227.3） x 109/L and （1 724.5 ± 316.3） x 109/L respectively, which was 5.4-fold and 7.1 -fold higher than that in the whole blood （P 〈 0.05）. The platelet concentration in auto-PRP gel stents produced by three centrifugations was higher than that in auto-PRP gel stents produced by two centrifugations（P 〈 0.05）. The number of ADSCs in auto-PRP gel stems produced by two or three centrifugations reached its peak which was （1.02 ± 0.13） x 104 and（1.00 ± 0.14） x 10n respectively when the platelet concentration was 500 x 109/L（P 〉 0.05）. The proliferation activity and the number of ADSCs in auto-PRP gel stents reached their peak when the platelet concentration was 500 x 109/L, and the TGF- [5 1 and collagen- II levels were significanlly higher when the platelet concentration was 500 x 109/L than when the platelet concentration was lower or higher than 500 x 109/L（P 〈 0.05）. Conclusion The detection rate of platelets is higher in auto-PRP gel stents produced by three centrifugations than by two centrifugations. However, no significant difference can be found in promoting proliferation of ADSCs in auto-PRP gel stents produced by two or three centrifugations. The proliferation and activity of ADSCs reach their peak when the platelet concentration is 500 x 109/L in auto-PRP gel stents, which maybe the optimal concentration for preparation of PRP.