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ID:32856568
大小:1.87 MB
页数:58页
时间:2019-02-16
《雌激素通过mapk信号通路刺激内皮祖细胞的增值和迁移》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、中图分类号:硕士学位论文雌激素通过MAPK信号通路刺激内皮祖细胞的增值和迁移院(系)、所临床医学院研究生姓名尹华曦学科、专业内科学导师姓名刘应才教授1目录1雌激素通过MAPK信号通路刺激内皮祖细胞增值和迁移„„„„„„„„„„„„„„„„„„„„„„„11.1中文摘要„„„„„„„„„„„„„„„„„„„„11.2英文摘要„„„„„„„„„„„„„„„„„„„„51.3前言„„„„„„„„„„„„„„„„„„„„„„101.4材料与方法„„„„„„„„„„„„„„„„„„„131.5结果„„„„„
2、„„„„„„„„„„„„„„„„„191.6讨论„„„„„„„„„„„„„„„„„„„„„„291.7结论„„„„„„„„„„„„„„„„„„„„„„381.8参考文献„„„„„„„„„„„„„„„„„„„„391.9英汉缩略词对照表„„„„„„„„„„„„„„„„462致谢„„„„„„„„„„„„„„„„„„„„„„473内皮祖细胞与MAPK信号传导通路(综述)„„„„„„„„„„„„„„„„„„„„„„„„482雌激素通过MAPK信号通路刺激内皮祖细胞的增值和迁移摘要目的:观察丝裂原活化蛋白激
3、酶(MAPK)及其亚族细胞外信号调节激酶(extracellular-signalregulatedproteinkinaseERK)、c-Jun氨基末端激酶(JNK)/应激激活蛋白激酶(SAPK)、p38在雌激素刺激内皮祖细胞(EPCs)迁移和增值中的作用。方法:(1)单个核细胞的分离:用50U/ml肝素2ml润湿空针后抽取脐带血约50ml,加入3%明胶16ml(脐带血:明胶=3:1)沉淀后加入人淋巴细胞分离液用密度梯度离心法分离出单个核细胞,活力计算活性达98%可以接种。(2)诱导分化:成纤维
4、细胞多于24小时内贴壁,因此培养24小时后取未62贴壁细胞以4×10/cm接种于包被有纤维连接蛋白的24孔培养板(每孔lml),每孔中加入M199培养液lml,添加10%胎牛血清、1%青链霉素(1万单位/ml)、VEGF(50ng/ml),置于5%CO2、饱和湿度37℃孵育箱中培养,换培养液培养至7d,贴壁细胞供实验用。(3)EPCs鉴定:培养细胞爬片后,然后分别加入鼠抗人CD133单克隆抗体(1:100稀释)、DiI-ac-LD、FITC-UEA-1分别在倒置相差显微镜及免疫荧光显微镜下观察拍照
5、。(4)实验分组:本实验共分五组,分别为对照组:正常培养EPCs未加入任何药物;雌二醇(E2)组:EPCs培养7天后加入E2;PD98,059(ERK抑制剂)组:培养7天后先用PD98,059作用细胞24h再加入E2;SP600125(JNK抑制剂)组:培1养7天后先用SP600125作用细胞24h再加入E2;SB203580(p38抑制剂)组:培养7天后先用SB203580作用细胞24h再加入E2。(5)观测指标:①酶联免疫检测仪检测各组活细胞数(结果以吸光值表示)。②选取特定浓度用流式细胞检测
6、仪分析各组细胞周期变化。③将细胞悬液种在Traswell小室上层,下层加入趋化因素,观察由上层小室迁入下层小室的细胞数。结果:(1)单个核细胞分离:从脐带血中分离的单个核细胞在显微镜下呈圆形、透亮,台盼蓝染色显示存活率>95%。(2)EPCs鉴定:取培养第7天的细胞做免疫荧光检测CD133呈绿色,阳性率达95%以上。取培养第9天的细胞检测内皮细胞特异性抗体Dil-ac-LDL呈红色,FITC-UAE-1呈绿色,Dil-ac-LDL和FITC-UAE-1双染的细胞为EPCs呈现黄色。染色结果显示黄色
7、细胞>90%。(3)酶联免疫检测活细胞数实验结果:对照组的吸光值为0.4163±0.0503,E2组吸光值为0.6940±0.0143,与对照组相比有显著意义(P<0.05),说明E2可以引起EPCs增值;PD98,059在5umol/L、10umol/L、20umol/L、40umol/L、80umol/L吸光值分别为0.7406±0.0376、0.6177±0.1043、0.5211±0.0793、0.5032±0.0975、0.4522±0.0406,与E2组比较PD98,059在20umo
8、l/L、40umol/L、80umol/L时有统计学意义(P<0.05),表明PD98,059可以阻断E2引起的EPCs增值;SP600125在1umol/L、25umol/L、50umol/L、75umol/L、100umol/L吸光值分别为0.6152±0.0406、0.4255±0.0395、0.3882±0.0185、0.4090±0.0761、0.4168±0.1012,与E2组相比SP600125在25umol/L、50umol/L、75umol/L、100umol/L
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