Maxadilan对人脂肪干细胞的影响

目的:探讨垂体腺苷酸环化酶激活肽Ⅰ型受体(PAC1受体)特异激动剂maxadilan对人脂肪干细胞(adipose-derived stem cells,ASCs)的增殖、凋亡和分化潜能的作用。方法:取人脂肪组织通过酶消化法分离培养ASCs。流式细胞术鉴定ASCs表面标志物,并进行ASCs向成骨成脂定向诱导。CCK-8法和流式细胞术检测maxadilan对ASCs活性的影响。采用波长为254 nm的紫外线(ultraviolet C,UVC)照射ASCs,CCK-8法测定不同剂量的UVC诱导ASCs凋亡后的吸光度。选择剂量为702 J/m2的UVC照射ASCs 24 h后,用流式细胞术和caspase 3和caspase 9试剂盒检测maxadilan对ASCs凋亡的影响。结果:流式细胞术检测表明细胞CD29、CD44、CD59和CD105表面抗原阳性,证实所提取的细胞是ASCs。CCK-8法检测发现80 nmol/L浓度的maxadilan对ASCs促增殖作用最强,流式细胞术分析也证实80 nmol/L maxadilan处理显著促进ASCs的增殖,与对照组比较差异有统计学意义(P0.05)。与仅被702 J/m2UVC照射的ASCs比较,80 nmol/L maxadilan显著抑制同等剂量UVC照射ASCs所诱导的、与caspase 3和caspase 9活性相关的细胞凋亡,2组比较差异有统计学意义(P0.05)。同时,2组细胞成骨和成脂的诱导分化均为阳性。结论:Maxadilan促进ASCs增殖,抑制ASCs凋亡,且不改变细胞向成骨和成脂诱导分化的潜能。Maxadilan有利于ASCs的体外生长与扩增。

Influence of maxadilan on human adipose-derived stem cells

AIM: To investigate the effect of maxadilan, which specifically activates pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells (ASCs). METHODS: ASCs from human adipose tissue were isolated by enzymatic digestion and cultured. ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry (FCM) and adipogenic/osteogenic induction. The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM. ASCs were irradiated by ultraviolet C (UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay. ASCs were exposed to 702 J/m² UVC for 24 h to induce apoptosis. The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels. RESULTS: Adipose-derived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM. The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation. The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimental group markedly improved the proliferation capacity of the cells compared with control group (P<0.05). The apoptosis of ASCs exposed to 702 J/m² UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L). Such process involved the caspase signaling pathway including caspase 3 and caspase 9. There was statistical significance (P<0.05) between experiment group (ASCs irradiated by UVC and supplemented with maxadilan) and control group (ASCs only irradiated by UVC). Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group. CONCLUSION: Maxadilan promotes proliferation and inhibits apoptosis of the ASCs. The differentiation potential of ASCs toward adipogenic and osteogenic lineages wouldn't be altered by maxadilan. Maxadilan would benefit to growth and expansion of ASCs in vitro.