福安泰-03对人脐静脉内皮细胞凋亡和小鼠创伤愈合的影响

研究福安泰-03（Fuantai，FAT-03）对人脐静脉血管内皮细胞（humanumbilicalveinen－dothelialcells，HUVECs）凋亡和小鼠创伤愈合的影响。MTT法检查FAT-03对HUVECs和人低分化鼻咽癌细胞（CNE－2Z）生长的影响：聚碳酸酯膜小室趋化运动模型（Transwellmodel）检测,]FAT-03对HU-VECs运动能力的影响；荧光显微镜观察FAT-03作用下HUVECs的形态变化；膜联蛋白V-异硫氰酸荧光素（AnnexinV-fluoresceinisothiocyanate，AnnexinV-FITC）双染检测Ⅳm03对HUVECs早期凋亡的影响；流式细胞术分析FAT-03对HUVECs周期及凋亡的影响；Westernblot法分析FAT-03对HUVECs的血管内皮细胞生长因子（VEGF）、Bcl．2、Bax表达的影响；小鼠背部创伤模型检查FAT-03对组织修复的影响；免疫组化法检查FAT-03对创伤组织微血管密度（microvesseldensity,MVD）和VEGF表达的影响。结果显示，FAT-03明显抑制HUVECs细胞的增殖和迁移，其抑制效果与剂量和作用时间相关，作用HUVECs24，48，72h的Ic50值为0．22，0．17，0．09mg／mL，但FAT-03对CNE．2Z细胞的生长却无明显的影响；0．16mg／mLFAT-03作用HUVECs24h对细胞迁移的抑制率为57．9％（P＜0．01）：FAT_03处理HUVECs48h，细胞的早期凋亡率增加（P〈0．05）；FAT-03阻滞HUVECs于G0／Gl期，并呈现典型的凋亡峰；0．16mg／mLFAT-03作用48，72h，HUVECs的凋亡率分别为14．6％、41．7％：鲋m03下调HUVECs的VEGF和抑凋亡基因Bcl-2的表达，上调促凋亡基因Bax的表达，其效果与剂量相关。FAT-03明显延迟小鼠创伤的愈合，且其作用与剂量相关。FAT-03组小鼠创伤周围组织微血管密度和VEGF阳性表达细胞都明显减少。因此，可以推测，FAT-03抑制HUVECs增殖并诱导其凋亡；抑制创伤组织的血管生成，进而延迟创伤愈合；它的这些作用可能与其下调VEGF、Bcl-2的表达，上调Bax的表达相关。

Effects of Fuantai-03 Isolated from Dasyatis akajei on the Apoptosis of Human Umbilical Vein Endothelial Cells and Mouse Wound Healing

The present study was undertaken to investigate the effects of Fuantai-03 （FAT-03） isolated from Dasyat akajei effected on the apoptosis of human umbilical vein endothelial cells and wound healing. MTT assay was performed to measure the effect of FAT-03 on cell growth; migration assay was performed using a Tran- swell model with polycarbonate membrane; apoptotic induction was determined by fluorescence microscopy and flow cytometry; Western blot analysis was performed for examing expressions of vascular endothelial growth factor （VEGF）, Bcl-2 and Bax. Mouse wound model was applied to investigate the effect of FAT-03 on wound healing; immunohistochemical staining assay was adopted to examine the microvessel density （MVD） and expression of VEGF in wound tissues. FAT-03 obviously inhibited proliferation and migration of HUVECs in a dose- and time- dependent manner the values of IC50 for the effect of FAT-03 on HUVECs at 24, 48, 72 h are 0.22 mg/mL, 0.17 mg/mL, 0.09 mg/mL, respectively, but FAT-03 did not show significant effect on the growth of human nasopharyngeal car- cinoma cell line （CNE-2Z）. 0.16 mg/mL FAT-03 decreased the percentage of migrating HUVECs at 24 h by 57.9% （P〈0.01）. FAT-03-treated HUVECs showed typical morphologic and cellular evidences of apoptosis. The expres- sions of VEGF and Bcl-2 in the FAT-03-treated HUVECs were evidently down-regulated, and the expression of Bax was obviously up-regulated. FAT-03 markedly decreased the MVD （P〈0.05） and down-regulated the expression of VEGF in mouse wound tissues, and inhibited tissue repairing. These findings provide evidences that FAT-03 signifi- cantly inhibits the proliferation and migration of HUVECs and induces their apoptosis, and inhibits tissue repairing in mouse wound model. The effects of FAT-03 might result from the down-regulation of expressions of VEGF and Bcl-2 and up-regulation of expression of Bax.