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低氧对胎盘基蜕膜间充质干细胞增殖、凋亡和血管内皮细胞生长因子表达的影响

黄永灿, 陈晓禾, 王佳, 李秀群, 解慧琪, 唐莉, 邓力

四川大学华西药学院.四川,成都 610041；四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室.四川,成都 610041

摘要

该文旨在探讨生理低氧和无血清培养条件下人胎盘基蜕膜间充质干细胞(placental decidua basalis--mesenchymal stem cells,PDB--MSCs)的生物学特征和细胞因子的表达情况。采用密度梯度离心法培养获得PDB--MSCs,利用MTT法、流式细胞术检测PDB--MSCs在不同氧浓度(20% O_(2)和1% O_(2))、有无血清(10% FBS和0% FBS)条件下各个时间点(6h、12h、24h、48h、72h、96h)的增殖和凋亡情况,并采用酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测无血清条件下各个时间点细胞上清液中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)含量。结果显示,在特定的时间内低氧可以促进PDB--MSCs增殖({sl P}<0.01,{sl n}=3),而血清对PDB--MSCs增殖的影响与氧浓度关系密切;同时,在本实验条件下,低氧、无血清分别或联合培养不会导致PDB--MSCs凋亡(sl P}>0.05,{sl n}=3);在无血清条件下,24h时低氧组PDB--MSCs表达较高水平的VEGF。以上结果提示,PDB--MSCs可能成为缺血相关组织工程产品种子细胞的一个新来源。

关键词： 基蜕膜; 间充质干细胞; 低氧; 增殖; 凋亡; 血管内皮细胞生长因子

Effects of hypoxia on human placental decidua basalis--mesenchymal stem cells proliferation,apoptosis and VEGF expression

Huang Yongcan, Chen Xiaohe, Wang Jia, Li Xiuqun, Xie Huiqi, Tang Li, Deng Li

West China School of Pharmacy,Sichuan University.Chengdu 610041,Sichuan；China

Abstract

Human placental decidua basalis-mesenchymal stem cells(PDB-MSCs) are multipotent cells from the human term placenta,which are ethically conducive,easily accessible and high-yielding source.PDB-MSCs can differentiate into adipogenic,osteogenic and neurogenic cells under appropriate conditions,which may be an attractive and alternative source of seed cells for tissue engineering.To investigate the effect of hypoxia(1% O_(2)) on human PDB-MSCs and the expression of cytokine,PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose(DMEM-HG) containing 10% fetal bovine serum(FBS),and the fifth passage of PDB-MSCs were taken.PDB-MSCs were divided into 4 groups according to the concentrations of O_(2) and FBS:20% O_(2),10% FBS;20% O_(2),0% FBS;1% O_(2),10% FBS;1% O_(2),0% FBS.The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h,12 h,24 h,48 h,72 h,and 96 h,respectively.Vascular endothelial growth factor(VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay(ELISA) at the same time points.The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS,while at 24 h under the condition of 0% FBS(P<0.01,n=3).In normoxia,the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS.However,in hypoxia,the number of cells cultured in 0% FBS(serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively(P<0.05,P<0.01,n=3).With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation,we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis(P>0.05,n=3).This conclusion may relate to the expression of VEGF which needs further research.In conclusion,the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation,suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.

Key words: decidua basalis;mesenchymal stem cells;Hypoxia;Proliferation;Apoptosis;vascular endothelial growth factor

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引用本文：

黄永灿, 陈晓禾, 王佳, 李秀群, 解慧琪, 唐莉, 邓力. 低氧对胎盘基蜕膜间充质干细胞增殖、凋亡和血管内皮细胞生长因子表达的影响[J]. 生理学报 2008; 60 (6): 783-789.

Huang Yongcan, Chen Xiaohe, Wang Jia, Li Xiuqun, Xie Huiqi, Tang Li, Deng Li. Effects of hypoxia on human placental decidua basalis--mesenchymal stem cells proliferation,apoptosis and VEGF expression. Acta Physiol Sin 2008; 60 (6): 783-789 (in Chinese with English abstract).