CAJ | 학술논문

目的从基因水平探究氧化应激在内皮细胞功能障碍中的作用及机制.方法用胶原酶消化结合免疫磁珠分选法纯化大鼠海绵体内皮细胞,通过黄嘌呤/黄嘌呤氧化酶(200μM/L,60mU/L,X/XO)体系氧化损伤48h,应用基因芯片研究损伤前后基因表达的差异,并利用分析软件筛选出与内皮细胞功能障碍相关的细胞信号通路.结果经纯化的海绵体内皮细胞纯度达96%(抗 vWF 法)和92.7%(流式细胞仪法)且生长增殖良好.氧化损伤后,内皮细胞透亮度减低,发生皱缩,释放乳酸脱氢酶(3200±223U/L vs 420±56U/L)显著增加(P <0.05).甲醛变性凝胶电泳中,提取的 RNA 在氧化损伤组与对照组均有清晰的18s、28s 条带电泳图,28s:18s 约为2:1.在对照组,表达基因13090个(占基因总数的31.92%),不表达27918个(占总数68.07%);氧化损伤组表达基因12039个(29.35%),不表达28972个(70.65%),其中,差异表达基因2480个(差异倍数2.0),1454个基因表达上调,1026个基因表达下调,并筛选出黏附功能、内吞作用、ErbB、细胞骨架肌动蛋白调控、蛋白转运等多个与内皮功能障碍关系密切的主要信号路径蛋白.结论氧化应激在内皮细胞功能障碍中有重要作用,并且通过多条信号通路调控相关基因的表达水平以影响内皮细胞功能.
목적종기인수평탐구양화응격재내피세포공능장애중적작용급궤제.방법용효원매소화결합면역자주분선법순화대서해면체내피세포,통과황표령/황표령양화매(200μM/L,60mU/L,X/XO)체계양화손상48h,응용기인심편연구손상전후기인표체적차이,병이용분석연건사선출여내피세포공능장애상관적세포신호통로.결과경순화적해면체내피세포순도체96%(항 vWF 법)화92.7%(류식세포의법)차생장증식량호.양화손상후,내피세포투량도감저,발생추축,석방유산탈경매(3200±223U/L vs 420±56U/L)현저증가(P <0.05).갑철변성응효전영중,제취적 RNA 재양화손상조여대조조균유청석적18s、28s 조대전영도,28s:18s 약위2:1.재대조조,표체기인13090개(점기인총수적31.92%),불표체27918개(점총수68.07%);양화손상조표체기인12039개(29.35%),불표체28972개(70.65%),기중,차이표체기인2480개(차이배수2.0),1454개기인표체상조,1026개기인표체하조,병사선출점부공능、내탄작용、ErbB、세포골가기동단백조공、단백전운등다개여내피공능장애관계밀절적주요신호로경단백.결론양화응격재내피세포공능장애중유중요작용,병차통과다조신호통로조공상관기인적표체수평이영향내피세포공능.
Objective To investigate the role of oxidative stress in endothelial dysfunction in gene level. Methods The collection of purified cavernosum endothelial cells were procedured by collagenase digestion combined with magnetic activated cell sorting(MACS), after injured by xanthine (200 μM/L) and xanthine oxidase (60mU/L, X/XO) for 48h, the differentially expressed genes and their pathways related to oxidative stress had been screened by gene chip and analysed by SAS software. Results The purity of endothelial cells attained 96%(anti-vWF) and 92.7%(FC), respectively, and the cells grew well. After injured by X/XO, the cells shrunk and their transparent ability reduced. Furthermore, the releasing of LDH of injuried cells (3200±223)U/L significantly increased than that of normal control group (420±56)U/L, P < 0.05. In formaldehyde- agarose gel electrophoresis, the intact bands(18s,28s) were showed in both groups, and 28s:18s were around 2:1. And in gene microarray, there were 13 090 (31.92% of total) genes expressing in normal control group with 27918 (68.07% of total) genes being not; in injured group, there were 12 039 (29.35%) genes expressing and 28972 (70.65%) genes did not. When the cut-off value was set as 2, there were 2 480 genes differentially expressed between X/XO injured group and normal control group, furthermore, 1 454 genes upregulated and 1026 genes downregulated. What’s more, relevant proteins of certain pathways, such as Adhension juncion, Endocytosis, ErbB signaling pathways, Regulation of actin cytoskeleton and Protein export had been pro-posed to be significant in endothelial dysfunction. Conclusion Oxidative stress may play a crucial role in endothelial dysfunction through some certain cell signal pathways by regulating the expression of oxidative stress-related genes.