缺氧对hBMSCs和胎盘来源MSCs增殖的影响

EFFECTS OF HYPOXIA ON PROLIFERATION OF hBMSCs AND HUMAN PLACENTAL DECIDUA BASALIS-MSCs

目的比较hBMSCs和人胎盘基蜕膜MSCs(human placental decidua basalis-MSCs,hPDB-MSCs)在化学缺氧条件下的增殖情况,为寻找组织工程新的种子细胞提供理论依据。方法采用密度梯度离心法分离培养hBMSCs与hPDB-MSCs,流式细胞仪检测细胞表面标志物;CoCl2建立化学缺氧模型,MTT法检测两种细胞在不同CoC12浓度(0、50、75、100、125、150、175、200μmol/L)和不同时间(6、12、24、48、72和96 h)的增殖情况。结果流式细胞仪检测显示hPDB-MSCs与hBMSCs均表达CD9、CD29、CD44、CD105、CD106、人类白细胞抗原ABC(human leucocyte antigen ABC,HLA-ABC),不表达CD34、CD40L和HLA-DR;但与hBMSCs相比,hPDB-MSCs阶段特异表达的胚胎抗原1(stage-specifi c embryonic antigen 1,SSEA-1)、SSEA-3、SSEA-4、肿瘤排斥抗原(tumor rejection autigen,TRA)-1-60、TRA-1-81表达更高。化学缺氧12 h内,hBMSCs与hPDB-MSCs增殖均被抑制,12 h后均能促进增殖;与对照组比较,hBMSCs经150μmol/L CoCl2作用24 h出现显著增殖(P<0.05),hPDB-MSCs在75μmol/L CoCl2作用12 h后出现显著增殖(P<0.05)。结论与hBMSCs相比,hPDB-MSCs表达更高的胚胎干细胞特有表面抗原,同时对化学缺氧所致的增殖影响更为敏感,可能成为一种新的组织工程种子细胞来源。

Objective To study the effect of hypoxia on the proliferation of hBMSCs and human placental decidua basalis-MSCs （hPDB-MSCs）, and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs, flow cytometry （FCM） was applied to detect cell surface marker. After establishing the experimental model of COCl2 chemical hypoxia, MTT method was applied to evaluate the proliferation of hBMSCs and hPDB-MSCs at different time points （6, 12, 24, 48, 72, 96 hours） with various COC12 concentration （0, 50, 75, 100, 125, 150, 175, 200 umol/L）. Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC （HLA-ABC）, but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 （SSEA-1）, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The proliferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably proliferated 24 hours after hypoxia with COC12 concentration of 150 lamol/L （P 〈 0.05）, while hPDB-MSCs were significantly proliferated 12 hours after hypoxia with COC12 concentration of 75 umol/L （P 〈 0.05）. Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the proliferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.