摘要
目的:观察两种尿毒症毒素同型半胱氨酸和晚期糖基化终产物是否协同影响血管内皮细胞的凋亡程度。方法:实验于2006-01/03在中南大学湘雅三医院中心实验室完成。①参考Makita的报道体外制备晚期糖基化终产物,将牛血清白蛋白50g/L、D-葡萄糖0.5mol/L、磷酸盐缓冲盐液0.2mol/L(pH7.4)和蛋白酶抑制剂用0.22μm滤膜过滤除菌,37℃孵育60d。采用荧光分光光度分析法鉴定。晚期糖基化终产物修饰的牛血清白蛋白中晚期糖基化终产物含量为90.52U/mg。②将不同浓度的同型半胱氨酸和晚期糖基化终产物单独或联合作用于人脐静脉内皮细胞株ECV304细胞,分为空白对照组;同型半胱氨酸干预组(分别加入0.05,0.1,0.5,1.0,5.0mmol/L的同型半胱氨酸);同型半胱氨酸+晚期糖基化终产物联合干预组(分别加入0.05,0.1,0.5,1.0,5.0mmol/L的同型半胱氨酸和终浓度为50mg/L的晚期糖基化终产物)。③采用Hoechst染色和流式细胞技术检测细胞凋亡程度。结果:①Hoechst33258染色检测内皮细胞凋亡结果:同型半胱氨酸+晚期糖基化终产物联合干预组在0.05～5.0mmol/L各个浓度较对照组和单纯同型半胱氨酸组都有明显差别(P<0.01)。②流式细胞仪检测内皮细胞凋亡结果:联合干预组总凋亡率为(15.31±2.14)%,早期凋亡率为(9.16±1.38)%,均显著高于同型半胱氨酸或晚期糖基化终产物单独干预组(P<0.01)。结论:同型半胱氨酸和晚期糖基化终产物对血管内皮细胞的凋亡具有协同诱导作用。
AIM： To investigate whether homocysteine acid （HCA） and advanced glycation end products （AGEs） have co-effects on the apoptosis of vascular endothelial cells.
METHODS： The experiment was accomplished at the Central Laboratory of Xiangya Third Hospital Affiliated to Central-South University from January to March 2006. （1） AGEs were prepared in vitro by referring to Makita＇s reports. Bovine serum albumin （BSA） （50 g/L）, D-glucose （0.5 mol/L）, phosphate buffer salt, solution （0,2 mol/L, pH 7.4） and proteinase inhibitor were sterilized by filtration with 0.2 μm of filter membrane, and then incubated at 37 % for 60 days. Samples were identified with fluorescence spectrophotometric method, and the content of AGEs modified BSA.was 90.52 U/mg. （2） ECV304 call line, derived from human umbilical vein endothelial calls, were cultured with HCA and AGE modified BSA respectively and both together, and then divided into blank control group, HCA group （0.05, 0.1, 0.5, 1.0, and 5.0 mmol/L of HCA was a added respectively）, and HCA ＋ AGE group （0.05, 0.1, 0,5, 1.0 and 5.0mmol/L HCA and 50 mg/L of AGE were added differently）. （3） The ratio of apoptosis was assessed by Hoechst staining and flow cytometry techniques.
RESULTS： （1） Hoechst33258 staining showed that the apoptotic ratios of HCA ＋ AGE group （0.05-5.0 mmol/L） were obviously higher than those in the blank control group and HCA group （P 〈 0.01 ）. （2） Flow cytometry techniques showed that the total apoptotic ratio of HCA ＋ AGE group was （15.31±2.14）%, and early apoptotic ratio was （9.16±1.38）%, which were significantly different from those in the HCA group and AGE group （both P〈 0.01 ）.
CONCLUSION：Both HCA and AGE can induce the apoptosis of vascular endothelial cells, and they have co-injury effects on these cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第6期1156-1158,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
湖南省自然科学基金资助项目(05JJ30059)~~
