摘要
研究福安泰-03(Fuantai,FAT-03)对人脐静脉血管内皮细胞(human umbilical vein en-dothelial cells,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(Transwell model)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(Annexin V-fluorescein isothiocyanate,Annexin V-FITC)双染检测FAT-03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;Western blot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(microvessel density,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HUVECs 24,48,72 h的IC50值为0.22,0.17,0.09 mg/mL,但FAT-03对CNE-2Z细胞的生长却无明显的影响;0.16 mg/mL FAT-03作用HUVECs 24 h对细胞迁移的抑制率为57.9%(P<0.01);FAT-03处理HUVECs 48 h,细胞的早期凋亡率增加(P<0.05);FAT-03阻滞HUVECs于G0/G1期,并呈现典型的凋亡峰;0.16 mg/mL FAT-03作用48,72 h,HUVECs的凋亡率分别为14.6%、41.7%;FAT-03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。
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.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2012年第4期332-342,共11页
Chinese Journal of Cell Biology
基金
"863"海洋技术领域专题(No.2007AA092422)
国家自然科学基金(No.30271493)
广东省自然科学基金重点项目(No.021386)
广东省海洋与渔业局科技兴海重大项目(No.A200099B01)资助项目~~
