【作者】 王石；

【导师】 陈晓虎；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：研究黄芪甲苷(astragaloside Ⅳ, AST)对EA-hy926细胞(一种人脐静脉内皮细胞)有无促增长作用,有无促进其形成新的血管的倾向,探讨这些作用是否通过ERK1/2信号转导通路实现。方法：1.黄芪甲苷对细胞增殖影响的体外实验细胞活性以CCK-8法评估。简而言之,EA-hy926细胞接种于96孔板,密度为2×104细胞每孔。预混的CCK-8和介质(10μl)加入96孔板,于37℃孵育细胞0.5-1h。用Beckman公司的3,550自动检测仪检测A450值。2.黄芪甲苷对血管内皮细胞管腔形成的体外实验我们以基质胶管腔形成实验来评估体外血管新生情况。将少生长因子基质胶置于96孔培养皿于37℃培养1小时。然后将1×104 EA-hy926细胞加入每孔,在基础培养基中加了10mg/L、30mg/L及100mg/L黄芪甲苷或二甲亚砜,另有一组在100mg/L黄芪甲苷的基础上同时用PD98059(10μM)处理30分钟,孵育4h,8h,12h。每孔计数5个区域。管腔的长度用Image-Pro Plus 6.0测量。3.黄芪甲苷促进血管新生的机制研究用批量生产指令法以Trizol-试剂分离总RNA。用实时PCR法测定VEGF的表达。实时PCR启始于以下PCR引物：forward:5’-CGGGAAACTGTGGCGTGAT-3’; reverse: 5’-CAAAGGTGGAGGAGTGGGT-3; VEGF:forward:5’-ATGAACTTTCTGCTGTCTTG-3’; reverse:5’-TGCATGGTGATGTTGGAC-3。使用SYBR-Green Universal Master Mix kit (ABI)来检测这些基因水平。每孔2×104细胞, 分别加入黄芪甲苷10mg/L,30 mg/L,100 mg/L于37度,5%二氧化碳浓度下培养特定的时间。提取全蛋白,每孔上样20μg细胞蛋白,于8-12% SDS-聚丙烯酰胺胶上电泳,电泳结束后,将蛋白转移至硝酸纤维素膜上。以5%脱脂奶粉(1XTBST配制)室温下封闭30分钟,孵育一抗(5%脱脂奶粉配制)过夜。以IX TBST冲洗膜3次,每次5分钟。孵育二抗,1X　TBST冲洗膜3次,每次5分钟,以ECL化学发光试剂检测其信号。结果：1.黄芪甲苷对细胞增殖影响的体外实验黄芪甲苷可促进EA-hy926细胞的增殖,其效应随黄芪甲苷的浓度增加而增加。本实验中100mg/L浓度的黄芪甲苷可达到最大的促细胞增殖作用。2.黄芪甲苷对血管内皮细胞管腔形成的体外实验我们在加入不同浓度黄芪甲苷的基质中发现有类毛细血管样管腔和完备的血管网络形成,并且在100mg/L浓度黄芪甲苷的实验组表现出最优的效果,同时对照组产生少量毛细血管样结构和不完全的网络,黄芪甲苷促进管腔形成的作用可被ERK1/2阻断剂PD98059所抑制。3.黄芪甲苷促进血管新生的机制研究在EA-hy926细胞中加入黄芪甲苷后,AST给药组较对照组P-ERK2蛋白表达明显上调,VEGF相关mRNA表达上调,VEGF蛋白表达也明显上调。当加入PD98059时,由于P-ERK2明显降低,ERK1/2信号通路被阻断,AST促血管内皮细胞增生作用和促管腔形成作用也在很大程度上被抑制。说明AST促血管新生作用是部分通过ERK1/2信号转导途径起作用的。结论：黄芪甲苷可通过激活ERKl/2信号转导通路调节血管内皮生长因子对EA-hy926细胞产生促进其增生及促进血管新生的作用。更多还原

【Abstract】 Purpose:This study aimed at determining the angiogenic effect of AST and its underlying mechanism.Methods:1.Cell Proliferation Assay in vitroViability of cells was assessed by Cell Counting Kit-8 (CCK-8) assay. In brief, EA-hy926 cells were seeded in a 96-well plates according to the density of 2×104 cells for each well. Premixed CCK-8 and medium (10μl) were added into 96-well plates, and cells were then incubated for 0.5-1h at 37℃. The values of A450 were obtained with the 3,550 automatic detector from Beckman.2. Tube Formation Assay in vitroA Matrigel tube-formation assay was also performed to assess in vitro angiogenesis. Growth factor-reduced Matrigel (BD) was placed in 96-well culture plates and allowed to set at 37℃ for 1 h. Then 1×104 EA-hy926 cells were added to each well and incubated in basic medium with 10mg/L AST,30mg/L AST, 100mg/L AST or DMSO,another group add 100mg/L AS-Ⅳ and PD98059 for 4h,8h,12h. Five fields were counted for each well. The length of the tube was measured by Image-Pro Plus 6.0.3. mechanism studyThe total RNA was isolated using Trizol-reagent according to manufacture’s instructions. Real-time PCR was used to measure the expression of VEGF. Real-time PCR was performed with the following PCR primers:GAPDH, forward 5’-CGGGAAACTGTGGCGTGAT-3’and reverse 5’-CAAAGGTGGAGGAGTGGGT-3; VEGF, forward 5’-ATGAACTTTCTGCTGTCTTG-3’and reverse 5’-TGCATGGTGATGTTGGAC- 3. SYBR-Green Universal Master Mix kit (ABI) were empoyed to detect the levels of these genes.Each well, with2×104 cells/well, was treated with AST 10mg/L,30 mg/L,100 mg/L respectively for the indicated duration at 5% CO2/95% room air and 37℃ for 24h. Protein of 20 μg/well was separated on 8-12% SDS-polyacrylamide gels and transferred onto nitro-cellulose membranes. After blocking in 1X TBST,5% nonfat dry milk at room temperature for 30 min, the membranes were incubated with the primary antibodies in blocking buffer (1X TBST,5% nonfat dry milk) overnight at 4℃. The membranes were washed three times for 5 min with 1X TBST and then incubated with secondary antibodies. After the membranes were washed three times for 5 min with 1X TBST, the signals were detected using ECL chemiluminescence reagents.Outcome:Our data revealed that AST promoted cell proliferation, and tube formation. Mechanism studies revealed that AST activated the extracellular signal regulated protein kinases 1/2(ERKl/2) pathway. All angiogenic effects of ASTwere reversed by ERK1/2 inhibitor (PD98059)Collectively AS-IV has a certain role in promoting angiogenesis in EA-hy926 cells, its mechanism of action involve in the activation of ERK1/2 cell signaling pathways.更多还原