羊水干细胞分泌细胞因子及其对人脐静脉内皮细胞增殖和凋亡的影响

Effects of human amniotic fluid stem cells on cytokines secretion and on endothelial cells proliferation and apoptosis

背景:关于细胞移植的功效,重点在于其分泌的促增殖或抗凋亡的细胞因子,羊水干细胞能否分泌相关细胞因子有必要深入分析。目的:观察分离培养的羊水干细胞分泌细胞因子情况,及其对人脐静脉内皮细胞增殖和凋亡的作用。设计、时间及地点:细胞学体外观察,于2008-12/2009-06在南昌大学第一附属医院高血压病研究所完成。材料:10例剖宫产孕妇足月羊水,50mL/例,用于分离培养羊水干细胞,同时留取其产后脐静脉用于分离培养内皮细胞,取前均获孕妇知情同意。方法:①贴壁法分离培养羊水干细胞,达80%融合时胰酶消化传代,取传至第3代细胞,调整细胞密度为5×108L-1,分为2组:缺氧羊水干细胞组通入体积分数为2%O2+体积分数为5%CO2+体积分数为93%N2,常规羊水干细胞组通入体积分数为5%CO2+体积分数为95%空气,两组细胞37℃培养24h,收集各自培养上清液及细胞以备分析。②取体外分离培养的第2代人脐静脉内皮细胞,以2×104细胞/孔的密度接种于12孔板,分为3组:对照组加入含体积分数为5%胎牛血清的EBM-2新鲜培养液2mL,常规羊水干细胞组加入含体积分数为5%胎牛血清的EBM-2新鲜培养液1mL+羊水干细胞培养液1mL,缺氧羊水干细胞组加入含体积分数为5%胎牛血清的EBM-2新鲜培养液1mL+缺氧诱导羊水干细胞培养液1mL,培养3d,加入10μg/L肿瘤坏死因子α诱导细胞凋亡。主要观察指标:流式细胞仪测定羊水干细胞表面抗原表达,ELISA法测定羊水干细胞分泌血管内皮细胞生长因子、肝细胞生长因子情况,RT-PCR法测定细胞因子mRNA的表达,检测内皮细胞增殖率及凋亡率。结果:培养7d后羊水干细胞呈长梭形,以漩涡状、辐射状排列,第3代羊水干细胞呈CD29+,CD105+,CD34-。常规培养的羊水干细胞可分泌血管内皮细胞生长因子、肝细胞生长因子;缺氧诱导条件下血管内皮细胞生长因子水平明显增加(P<0.01),其mRNA表达明显上调(P<0.05),而肝细胞生长因子含量及mRNA的表达均无明显变化(P>0.05)。与对照组比较,培养3d后常规羊水干细胞组、缺氧羊水干细胞组内皮细胞数均明显增加(P<0.05),肿瘤坏死因子α诱导凋亡24h后内皮细胞凋亡率均明显降低(P<0.05),且缺氧羊水干细胞组变化幅度大于常规羊水干细胞组(P<0.05)。结论:羊水干细胞可分泌血管内皮细胞生长因子、肝细胞生长因子,羊水干细胞培养液能促进内皮细胞增殖,抑制内皮细胞凋亡。经缺氧处理后,羊水干细胞分泌血管内皮细胞生长因子的能力增加,对内皮细胞的增殖和凋亡干预作用增强。

BACKGROUND： The benefit of cell therapy may be partly due to the secretion of angiogenic and antiapoptotic growth factors. Whether amniotic fluid stem cells （AFS） could secrete some growth factors requires further studies. OBJECTIVE： To isolate and culture AFS cells, and explore the angiogenic or antiapoptotic effect of cytokines secreted by AFS on endothelial cells. DESIGN, TIME AND SETTING： A in vitro cytological experiment was performed at the Institute of Hypertensive Disease, First Affiliated Hospital, Nanchang University from December 2008 to June 2009. MATERIALS： Term amniotic fluid of ten samples, 50 mL/case, was obtained following caesarean delivery. The umbilical vein was used to isolate endothelial cells. Written informed content was obtained from all women. METHODS： AFS isolated from human amniotic fluid was cultured and digested by trypsin at confluence of 80%. The third passage of cells at a density of 5×10^8/L were divided into two groups： hypoxia group： the cells were cultured in 2% O2 ＋ 5% CO2 ＋ 93% N2; normal group： the cells were cultured in 5% CO2 ＋ 95% air. Two groups were cultured at 37 ℃ for 24 hours. The supematant of two groups was collected. The second passage of human umbilical vein endothelial cells cultured in vitro was collected and seeded onto 12-well culture plate at a density of 2×10^4 cells/well, and divided into 3 groups： control group was cultured in 2 mL EBM-2 containing 5% fetal bovine serum （FBS）; normal group was cultured in 1 mL EBM-2 containing 5% FBS and 1 mL AFS cell culture solution; hypoxia group was cultured in 1 mL EBM-2 containing 5% FBS and 1 mL hypoxia AFS cell culture solution for 3 days, followed by incubation with 10 μg/L tumor necrosis factor （TNF）-α. MAIN OUTCOME MEASURES： AFS surface phenotype was examined by flow cytometry; the secretion level and mRNA expression of vascular endothelial cell growth factor （VEGF） and hepatocyte growth factor （HGF） were examined by ELISA or RT-PCR. The proliferation and apoptotic rates of endothelial cells were examined. RESULTS： AFS cells were long fusiform-shaped and arranged radially after 7 days of culture. The third passage of AFS ceils expressed CD29 and CD105 while did not express CD34. AFS cells of normal culture secreted VEGF and HGF; AFS cells of hypoxia condition significantly increased secrete of VEGF （P 〈 0.01 ）, and VEGF mRNA expression was significantly upregulated （P 〈 0.05）, while HGF and mRNA expression remained unchanged （P 〉 0.05）. Compared with control group, the number of endothelial cells was significantly increased in normal and hypoxia AFS cell groups after 3 days of culture （P 〈 0.05）. After cocultured with TNF-α for 24 hours, the apoptosis rates of endothelial cells in AFS-conditioned medium was significantly decreased （P 〈 0.05）, and the change degree of hypoxia AFS cell group was greater than normal AFS cell group （P 〈 0.05）. CONCLUSION： AFS can secrete cytokines such as VEGF and HGF. Moreover, it significantly promotes endothelial cells proliferation and inhibits apoptosis. Under hypoxia condition, the secretion of VEGF from AFS cells is increased, and the effects on endothelial cells proliferation and apoptosis are enhanced.