鼠尾胶原联合血小板源性生长因子BB培养内皮细胞

Combination of rat tail collagen and platelet-derived growth factor-BB for culture of human umbilical vein endothelial cells

背景:有研究证实,Ⅰ型鼠尾胶原可促进成肌纤维细胞的增加,对内皮细胞的迁移及成管有较为明显的作用,推断胶原可为细胞的生长提供一个较为合适的内环境。目的:探讨鼠尾胶原联合血小板源性生长因子BB抗人脐静脉内皮细胞凋亡的效果及安全性。方法:将第4代人脐静脉内皮细胞培养于铺有鼠尾胶原的培养皿上,用Alamar Blue法检测不同时间点的还原比率。将第4代人脐静脉内皮细胞分为4组培养于24孔板,其中生长因子组是在细胞接种前加入血小板源性生长因子BB蛋白于细胞悬浮液中;联合组需事先加入血小板源性生长因子BB蛋白于鼠尾胶中,铺于板底;最后各组均使用H2O2诱导凋亡,73 h后采用Western blot测定血小板源性生长因子BB、凋亡相关蛋白及抗凋亡蛋白的表达,同时Tunnel检测细胞凋亡阳性率。结果与结论:在铺有鼠尾胶原培养皿中培养人脐静脉内皮细胞的成管数量明显多于正常培养人脐静脉内皮细胞的成管数量(P<0.05)。鼠尾胶原培养的细胞与正常对照细胞生长状态相似,说明鼠尾胶原对细胞无明显毒性作用。联合组血小板源性生长因子BB、Bcl-2、p-Akt表达高于其余3组(P<0.05),Bax表达低于其余3组(P<0.05)。联合组细胞凋亡率低于生长因子组、H2O2组(P<0.05)。表明鼠尾胶原对人脐静脉内皮细胞无明显毒性作用,联合血小板源性生长因子BB生长因子后可显著增强抗细胞凋亡效果。

BACKGROUND：Rat tail colagen type I can promote the increase of muscle fiber cels and migration and tube formation of endothelial cels, which is speculated to provide a more suitable internal environment for the growth of cels. OBJECTIVE：To observe the effect and safety of platelet-derived growth factor-BB （PDGF-BB） with rat rail colagen against apoptosis of human umbilical vein endothelial celsin vitro. METHODS：The passage 4 human umbilical vein endothelial cels were cultured in the medium of rat rail colagen and the reduction ratio in different time points was detected by Alamar Blue. The passage 4 human umbilical vein endothelial cels were divided into four groups and cultured in 24-wel culture plates： control group, PDGF-BB group, H2O2 group, PDGF-BB＋colagen group. H2O2 was used to induce cel apoptosis in al the groups. Western blot was used to detect the expression of PDGF-BB, apoptosis-related protein and anti-apoptosis protein after 72 hours. Meanwhile, TUNEL method was used to detect cel apoptotic rate. RESULTS AND CONCLUSION：The tube formation in the human umbilical vein endothelial cels was more than that cultured in normal medium （P 〈 0.05）. Cels cultured with rat tail colagen showed similar growth to normal control cels, indicating rat tail colagen had no cytotoxicity. The expressions of PDGF-BB, Bcl-2, and p-Akt in the PDGF-BB＋colagen group were significantly higher than those in the other three groups （P 〈 0.05）, but the expression of Bax was lower than that in the other three groups （P 〈 0.05）. The apoptotic rate in the PDGF-BB＋colagen group was lower than the PDGF-BB group and H2O2 group （P 〈 0.05）. These findings indicate that rat tail colagen has no cytotoxicity to human umbilical vein endothelial cells, and rat tail collagen combined with PDGF-BB can predominantly enhance anti-apoptosis effects.