不同抗凝剂和激活剂组合在自体富血小板血浆凝胶支架促进脂肪干细胞增殖的影响

目的 探讨不同抗凝剂和激活剂组合在自体富血小板血浆(PRP) 凝胶支架对脂肪干细胞(ADSCs) 增殖的影响.方法 在制作自体富血小板血浆支架并植入ADSCs 过程中,使用不同抗凝剂(肝素、EDTA) 和激活剂(凝血酶、Ⅰ型胶原),分为EDTA 与凝血酶组,EDTA 与Ⅰ型胶原组,肝素与凝血酶组,肝素与Ⅰ型胶原组,并设置空白对照组.每日计数细胞并绘制细胞生长曲线,第2、3、4、6 天用MTT 比色法分析各组脂肪干细胞的存活和增殖能力,以ELISA 定量检测生长因子TGF-β1 及细胞的Ⅱ胶原.通过RT-PCR 测定sox-9 基因的表达.结果 与空白对照组相比,各组均保持较高的增殖活性(P<0.05),EDTA 与凝血酶组脂肪干细胞增殖计数最高.各组富血小板血浆均能明显促进生长因子TGF-β1 的释放和Ⅱ型胶原合成以及sox-9 基因的表达,其中EDTA 与凝血酶组生长因子TGF-β1 和Ⅱ胶原量以及sox-9 基因的表达最高.结论 EDTA 与凝血酶组自体富血小板血浆(PRP) 对促进脂肪干细胞增殖效果最好.

Effect of combined anticoagulants and agonists on proliferation of adipose derived stromal cells in auto-platelet rich plasma gel stent

Objective To study the effect of combined anticoagulants and agonists on proliferation of adipose derived stromal cells(ADSCs) in auto-platelet rich plasma(PRP) gel stent.Methods ADSC,implanted into PRP gel stent using different anticoagulants(heparin,EDTA) and agonists(thrombin,collagen-1),were divided into EDTA and thrombin group,EDTA and collagen-1 group,thrombin and collagen-1 group,heparin and thrombin group,and control group.ADSC were counted everyday for 7 days and cell growth curve was plotted.Survival rate and proliferation activity of ADSC were analyzed by methyl thiazolyl tetrazolium(MTT) assay on day 2-4 and 6.Growth factor TGF-β1 and collagen-Ⅱ were detected by ELISA.Expression level of sox-9 in ADSC was measured by RT-PCR.Results The proliferation activity of ADSC was significantly higher in EDTA and thrombin group,EDTA and collagen-1 group,thrombin and collagen-1 group,heparin and thrombin group,especially in EDTA and thrombin group,than in control group(P<0.05).Auto-platelet rich plasma gel could significantly promote the release of TGF-β1,synthesis of collagen-Ⅱ,and expression of sox-9,especially in EDTA and thrombin group(P<0.05).Conclusion The effect of auto-platelet rich plasma gel is the best on proliferation of ADSC in EDTA and thrombin group.