高级蛋白氧化产物对人脐静脉内皮细胞ECV-304的作用  

Effects of advanced oxidation protein products modified protein on human umbilical vein endothelial cells

作　　者：范虹[1] 丁峰[1] 顾勇[1] 朱秋毓[1] 林善锬[1] 

机构地区：[1]复旦大学附属华山医院肾脏科,上海200040

出　　处：《复旦学报（医学版）》2007年第2期176-182,共7页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(30300160);复旦大学985工程重点扶植学科计划资助项目

摘　　要：目的 观察体外制备的高级蛋白氧化产物修饰蛋白（AOPP-HSA）对人脐静脉内皮细胞（ECV-304细胞株）的作用.方法 在高压液相色谱仪（HPLC）中将人血清白蛋白（HSA）用次氯酸处理制备AOPP-HSA,观测AOPP-HSA对ECV-304细胞增殖和凋亡,细胞间黏附分子-1（ICAM-1）和E-选择素（E-selectin）及单核细胞趋化蛋白-1（MCP-1）的表达,以及P50、P65和I-кB的mRNA表达含量的影响.结果 （1）AOPP-HSA与ECV-304细胞共同孵育后,可使其增殖指数下降,约为对照组的70%～80%.凋亡试验结果：AOPP-HSA组细胞早期凋亡较对照组升高17%.（2）ECV-304细胞有ICAM-1和MCP-1的基础表达,与AOPP-HSA共同孵育24 h后,两者的表达均有上调,以VitE预先孵育后,其表达量均较相应浓度AOPP-HSA组有明显下降;ECV-304细胞无E-selectin的基础表达,给予AOPP-HSA后也无改变.（3）ECV-304细胞与AOPP-HSA共同孵育后,其P50、P65的mRNA的相对表达含量均显著上调,在VitE干预组,P50、P65的相对表达含量较相应AOPP-HSA组下降.I-кB mRNA的相对表达含量在AOPP-HSA 100～400 μmol/L各组均明显下降,而在AOPP-HSA 600 μmol/L组中,其I-кB的相对表达含量出现显著的升高,给予VitE干预后,I-кB的相对含量也出现明显升高.结论 AOPP-HSA对ECV-304细胞具有多种生物学效应,可抑制增殖、促进凋亡、上调黏附分子和趋化因子的表达.这些作用可能是通过影响NF-кB而产生的.氧化应激参与了这些作用的发生.Purpose The present study was protein on human endothelial cells. Methods conducted to investigate the effects of AOPP modified AOPP-human serum albumin（AOPP-HSA） was prepared by exposing human serum albumin to HOCl. Briefly,The AOPP-HSA preparation was incubated for 30 min at room temperature and then dialyzed overnight against PBS and tested for AOPP content. Human umbilical endothelial cells （ECV-304） were cultured in vitro. The proliferation of EC was determined by MTT colorimetric method and apoptosis was detected by Annexin-V-Fluos. The expression of adhesion molecule, intercellular adhesion molecule-1 （ICAM-1） and E-selectin was assessed by means of flow cytometry. The expression of monocyte chemoattractant protein-1 （MCP-1） in culture medium was assessed by ELISA. The mRNA expression of P50, P65, I-κB were semi-quantitated by reverse transcription-polymerase chain reaction （RT-PCR）. Results The results showed ICAM-1,but not E-selectin was constitutively expressed on EC. The expression of ICAM-1 and MCP-1 was enhanced by AOPP-HSA after a chosen incubation period of 24 h,while E-selectin was not observed. Vitamin E inhibited the expression of ICAM-1 and MCP-1 induced by AOPP-HSA. On cell cycle, AOPP-HSA inhibited the proliferation of endothelial cell by 70% - 80%, and induced apoptotic cell death. RT-PCR studies showed that the mRNA expressions of P50,P65 in the AOPP-HSA groups were greatly up-regulated. Administration of vitamin E reduced these gene expressions. On the expression of I-κB mRNA, AOPP-HSA had dual effect. In these AOPP-HSA 100 - 400 μmoL/L groups, the mRNA expression of I-κB was markedly declined. By contraries, in the AOPP-HSA 600 μmol/L group, the mRNA expression of I-κB was greatly up-regulated. Administration of vitamin E also increased the gene expression. Conclusions Taken together,these data indicate that AOPP-HSA,but not purified HSA,represents a novel class of proinflammatory mediators. AOPP-HSA up-regulate the expression of adhesion molecules and chemoattr

关 键 词：高级蛋白氧化产物 内皮细胞ECV-304 氧化应激 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]