平阳霉素作用静脉畸形内皮细胞后TGF-β1的表达

Pingyangmycin-regulated expression of transforming growth factor-β1 in human venous malformation endothelial cells

目的:研究平阳霉素(PYM)作用于体外原代的培养人静脉畸形内皮细胞后转化生长因子β1的表达。方法:通过体外原代培养人静脉畸形内皮细胞,采用ELISA和RT-PCR技术检测不同浓度PYM作用人静脉畸形内皮细胞TGF-β1表达。结果:ELISA发现内皮细胞在自然状态下能分泌TGF-β1,体外培养12h后,随着培养时间的增加,TGF-β1的释放量也随之增加。对照组内皮细胞培养48h后,细胞上清中TGF-β1含量达到(537.56±2.61)pg/ml。500ng/ml和1μg/ml PYM作用内皮细胞12h后,内皮细胞表达分泌的TGF-β1较高,且差异有统计学意义。其他浓度PYM作用于静脉畸形内皮细胞24、48h后,TGF-β1表达与对照组细胞差异无统计学意义。PYM浓度≥10μg/ml,内皮细胞TGF-β1表达量较低。RT-PCR结果显示,对照组(未加药物)细胞TGF-β1mRNA阳性表达,PYM浓度为500ng/ml和1μg/ml时TGF-β1mRNA表达较高,PYM浓度在10～100ng/ml之间,TGF-β1mRNA表达量与对照组之间差异无统计学意义。结论:内皮细胞在PYM致静脉畸形组织纤维化过程中能分泌促组织纤维化细胞因子TGF-β1促进组织纤维化。

Abstract Objective：To explore the role of human venous malformation endothelial cells in venous malforma- tion tissue fibrosis induced by intra-lesional injection with PYM, we examined PYM-regulated expressions of transforming growth factor-β1 （TGF-β1） in vitro in primary cultured human venous malformation endothelial cells （HVMECs）. Method： Expressions of TGF-β1 in supernate were studied in HVMECs stimulated with different con- eentrations（10 ng/ml～100/μg/ml） of PYM for different time interval（1 h, 6 h, 12 h, 24 h, 48 h） in vitro by means of Enzyme-Linked Immunosorbnent Assay（ELISA）. According to the result of ELISA, expression of＇TGF- β1 mRNA in HVMECs stimulated with different concentrations of PYM for 12 h was examined by RT-PCR. Re- suit：Expression of TGF-β1 in supernate of control cells was examined, after 12 h cultured, the amount of TGF-β1 increased along with the cell culture time. After 48h the amount was 538 pg/ml. After 12 h exposure to PYM at 500 ng/ml and 1 /μg/ml, an increased TGF-β1 expression was observed. A decreased TGF-β1 expression was found in HVMECs treated with 100/μg/ml of PYM. There were no significant differences in expressions of TGF- β1 in HVMECs treated with different concentrations（≤10 /μg/ml） bf PYM for the same time interval. RT-PCR revealed positive expression of mRNA of TGF-β1 in control group. After 12 h exposure to PYM at 500 ng/ml and 1μg/ml, a significant increased TGF-β1 mRNA expression was observed. There was no significant difference in TGF-β1 mRNA expression in HVMECs treated with 10 -100 ng/ml PYM. Conclusion： These findings indicated that HVMECs were stimulated to secrete TGF-β1 in the presence of special concentrations of PYM for special time interval. One of the mechanisms of sclerotherapy of venous malformation with PYM may be up-regulation of ex- pression of TGF-β1 in HVMECs, which play an important role in the process of tissue fibrosis.