摘要
目的建立2型糖尿病血瘀证的血管内皮细胞损伤模型。方法取对数生长期的正常人脐静脉内皮细胞(ECV-304),干预分组如下:空白对照组(无血清的DMEM)、健康组(健康人血清)、糖尿病血瘀证组(糖尿病血瘀证患者血清)和糖尿病非血瘀证组(糖尿病非血瘀证患者血清)。采用MTT法观察细胞活性;在倒置相差显微镜、扫描电镜和透射电镜下观察细胞形态的变化;应用放免法测定内皮素(ET)含量;硝酸还原酶法测定一氧化氮(NO)含量;利用双抗夹心ELISA法检测各组细胞培养上清液中内皮细胞蛋白C受体(EPCR)、血管内假性血友病因子(vWF)和血栓调节蛋白(sTM)的含量;应用激光扫描共聚焦显微镜,采用Fluo-3/AM作为荧光指示剂观察细胞内游离钙浓度的变化;并采用荧光探针标记的鬼笔环肽染色法观察细胞肌动蛋白微丝分布的差异。结果①10%浓度的糖尿病血瘀证组的OD值较健康组明显降低(P<0.001);②糖尿病血瘀证组的ET水平比空白对照组和糖尿病非血瘀证组升高(P<0.001);糖尿病血瘀证组的NO水平比空白对照组降低(P<0.001);但和糖尿病非血瘀证组相比差异没有统计学意义(P>0.05);③内皮细胞经过患者血清损伤后,糖尿病血瘀证组分泌的EPCR含量比空白对照组升高(P<0.001),但低于糖尿病非血瘀证组(P<0.001);糖尿病血瘀证组分泌的vWF和sTM含量比空白对照组升高(P<0.001),和糖尿病非血瘀证组相比差异没有统计学意义(P>0.05);④糖尿病血瘀证组胞浆内荧光分布不均匀,荧光强度高于正常对照组和糖尿病非血瘀证组(P<0.001);⑤空白对照组和健康组细胞骨架微丝分布多规则清晰,保持着细胞的正常外形,糖尿病血瘀证组的细胞骨架微丝断裂且排列紊乱,糖尿病非血瘀证组细胞微丝断裂但排列较为规则。结论应用2型糖尿病血瘀证患者血清干预ECV-304可以在体外表现出血管内皮细胞损伤的种种征象,初步建立一种病证结合的血瘀证细胞损伤模型。
Objective To establish the vascular endothelial cell lesion model of blood-stasis syndrome of type 2 diabetes mellitus.Methods ECV-304 ceils of logarithmic phase were intervened and divided into the blank control group（group 1）,health group（group 2）,blood-stasis syndrome of diabetes mellitus group（group 3） and diabetes mellitus group（group 4）.The activities of ECV-340 cells were observed by using MTT test.The morphological changes of the cells were observed by inverted phase contrast microscope,scanning electron microscope and transmission electron microscope.The levels of ET and NO were detected by using radioimmunoassay and nitrate reductase method respectively.The levels of endothelial protein C receptor（EPCR）,yon Willebrand factor（vWF） and thrombonodulin（sTM） were detected by applying double-antibodies-sandwich-ELISA method.The changes of free calcium in the cells were observed by laser scanning confocal microscope（LSCM） taken Fluo-3/AM as fluorescence indicator.The distribution of actin microfilament was observed by using rhodamine phalloidin staining. Results①The value of optical density（OD） decreased significantly in group 3 than that in group 2 （P〈0.001）.②In group 3 ET level was higher than that in group 1 and that in group 4（P〈0.001）, NO level was lower than that in group 1（P〈0.001） and had no statistical difference compared with that in group 4（P〉0.05）.③In group 3 EPCR level was higher than that in group 1 and lower than that in group 4（P〈0.001）,the level of vWF was higher than that in group 1（P〈0.001） and had no statistical difference compared with that in group 4（P〉0.05）,and sTM level was higher than that in group 1（P〈0.001） and had no statistical difference compared with that in group 4（P〉0.05）.④In group 3 the distribution of fluorescence showed uneven and fluorescence intensity was higher than that in group 1 and group 4（P〈0.001）.⑤In group 1 and 2 the distribution of microfilament cytoskeleton was regular and clear with normal cell shape,while in group 3 it was broken and irregular,and in group 4 it was broken but regular.Conclusion ECV-304 cells showed various signs of vascular endothelial cell lesion after the interference by the serum from the patient with type 2 diabetes mellitus.The vascular endothelial cell lesion model of blood-stasis syndrome combined disease and syndrome was established initially.
出处
《北京中医药大学学报》
CAS
CSCD
北大核心
2009年第11期742-746,754,F0003,共7页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(No.30572289)
广东省自然科学基金资助项目(No.5006019)
关键词
2型糖尿病
血瘀证
血管内皮细胞损伤
细胞模型
type 2 diabetes mellitus
blood-stasis syndrome
vascular endothelial cell lesion
cell model
