Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts
-
摘要: 目的 探讨人羊膜间充质干细胞(hAMSC)培养上清液(hAMSC-CS)对人Fb生物学功能的影响。 方法 (1)取废弃胎盘新鲜羊膜组织,分离hAMSC,并进行传代培养。倒置相差显微镜下观察培养3 d及第3代hAMSC形态。(2)取2批次第3代hAMSC,免疫荧光染色法检测细胞波形蛋白表达,流式细胞仪检测细胞表面标志物CD90、CD73、CD105及CD45表达。(3)收集培养72 h的第3代hAMSC-CS,ELISA法检测其中胰岛素样生长因子Ⅰ(IGF-Ⅰ)、血管内皮生长因子(VEGF)、EGF、bFGF的含量。(4)取包皮环切术后废弃包皮,分离人Fb,并进行传代培养。采用第3代人Fb进行以下实验。按随机数字表法(分组方法下同)将细胞分为空白对照组及10%、30%、50%、70%hAMSC-CS组,每组48孔。空白对照组用含体积分数2%FBS的DMEM/F12培养液培养,后4组分别用含相应体积分数hAMSC-CS和体积分数2%FBS的DMEM/F12培养液培养。培养12、24、48、72 h,采用细胞计数试剂盒8及酶标仪检测细胞增殖活性。选取使人Fb增殖活性最高的相应体积分数hAMSC-CS用于以下实验。(5)另取人Fb,分为空白对照组及50%hAMSC-CS组,同(4)处理,每组4孔。分别于划痕后0(即刻)、12、24、48、72 h,于倒置相差显微镜下观测细胞迁移距离。(6)另取人Fb,同(5)分组处理,每组3瓶,流式细胞仪检测细胞凋亡率。对数据行析因设计方差分析、重复测量方差分析、单因素方差分析、LSD检验、
t 检验。 结果 (1)培养3 d,多数hAMSC形态较大,呈纺锤形多突起的Fb样或扁平多角形。第3代hAMSC呈梭形或纺锤形,波形蛋白表达呈强阳性,细胞表面标志物CD90、CD73、CD105表达阳性,CD45表达阴性。(2)hAMSC-CS中IGF-Ⅰ、VEGF、EGF、bFGF的含量分别为(11.7±1.0)、(316±68)、(6.1±0.4)、(1.49±0.05)pg/mL。(3)培养12~72 h,各hAMSC-CS组人Fb增殖活性明显高于空白对照组(P 值均小于0.01),50%hAMSC-CS组人Fb增殖活性最高。(4)划痕后0 h,2组人Fb划痕宽度几乎相同。划痕后12~72 h,50%hAMSC-CS组人Fb迁移距离明显长于空白对照组(P 值均小于0.01)。(5)空白对照组人Fb凋亡率为(16.2±2.4)%,明显高于50%hAMSC-CS组的(7.4±3.6)%(t =6.710,P <0.01)。 结论 hAMSC-CS对人Fb的增殖和迁移具有促进作用,对人Fb的凋亡有抑制作用。Abstract: Objective To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. Methods (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, andt test. Results (1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (withP values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (withP values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%,t =6.710,P <0.01]. Conclusions hAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.
点击查看大图
计量
- 文章访问数: 49
- HTML全文浏览量: 3
- PDF下载量: 4
- 被引次数: 0