雌激素通过mapk信号通路刺激内皮祖细胞的增值和迁移

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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