摘要
背景:切应力可以直接介导内皮细胞表达一些编码与血管生成相关的细胞因子基因,血液的流体切应力对调节血管结构和功能具有重要的生物学作用。目的:观察流体层流切应力对人脑动静脉畸形血管内皮细胞增殖与原癌基因c-myc表达的影响。设计:随机对照的技术方法。单位:解放军沈阳军区总医院神经外科。对象:实验于2006-11/2007-02在解放军沈阳军区总医院全军神经医学研究所完成。选用2006解放军沈阳军区总医院神经外科SpetzlerⅡ-Ⅲ级20例脑动静脉畸形患者手术切除的人脑动静脉畸形新鲜标本,全部病例术前均经全脑血管造影证实。M199培养液(Gi1bco BRL),优质胎牛血清(HyClone),内皮细胞生长添加剂(ECGS;美国Sigma),CO2培养箱(美国Forma Scientific),细胞周期分析试剂盒(BD公司),流式细胞仪(FACS Calibur,BD公司),鼠抗人c-myc单克隆抗体(美国Santa Cruz公司),RT-PCR试剂盒(Promega)。方法:采用组织块贴壁法培养人脑动静脉畸形血管内皮细胞,按所受切应力大小将细胞分为4组:对照组、低切组、中切组和高切组。将培养的人脑动静脉畸形内皮细胞单层置于平板流动系统内,低切组、中切组和高切组细胞分别经低、中、高切应力作用8h,对照组切应力为0Pa。应用流式细胞术测定细胞增殖指数。检测c-myc蛋白表达。检测c-mycmRNA表达。主要观察指标:不同切应力作用下内皮细胞c-myc蛋白及mRNA表达和细胞增殖指数。结果:①细胞增殖指数:中切组和高切组内皮细胞增殖指数高于对照组(P<0.05,0.01)。②c-myc蛋白表达:c-myc免疫阳性表达随切应力增高而递增,各切应力组与对照组比较差异均有统计学意义(P<0.05～0.01)。③c-mycmRNA表达:低切组和中切组内皮细胞增殖指数高于对照组(P<0.05)。结论:流体层流切应力能够在基因转录水平诱导c-myc表达,并可能通过激活c-myc基因表达而促进人脑动静脉畸形血管内皮细胞增殖。
BACKGROUND：Shear stress can directly mediate the expression of endothelial calls, especially some cytokine genes whose codes are related to angiogenesis. Otherwise, flow shear stress of blood plays an importantly biological role in regulating vascular structure and function. OB3 ECTIVE： To observe the effects of laminar flow shear stress on the proliferation of vascular endothelial calls and the expression of protooncogene c-rnyc in human cerebral arteriovenous malformation. DESIGN： Randomized controlled study SETTING： Department of Neurosurgery, Shenyang General Hospital of Military Area Command of Chinese PLA. MATERIALS ： The experiment was carded out in the Neuromedical Institute, General Hospital of Shenyang Military Area Command of Chinese PLA from November 2006 to February 2007. Fresh samples of human cerebral arteriovenous malformation were derived from 20 patients who were of grade Spetzler Ⅱ-Ⅲ and received resection of human cerebral arteriovenous malformation in the Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA in 2006. All cases were diagnosed with whole-brain angiography before operation, The main reagents and equipments were detailed as follows： M199 culture media （Gilbco BRL）, quality fetal bovine serum （HyCIone）, endothelial cell growth supplement （ECGS; Sigma, USA）, CO2 incubator （Forma Scientific, USA）, flow cytometry analysis of cell cycle kit （BD Company）, flow cytometer （FACS Calibur, BD Company）, rat-anti-human c-myc monoclonal antibody （Santa Cruz Company, USA）, and reverse transcription polymerase chain reaction （RT-PCR） kit （Promega）. METHODS： Tissue explants adherent method was used to culture vascular endothelial cells of human cerebral arteriovenous malformation, and then the calls were classified into 4 groups based on degree of shear stress, including control group, low shear stress group, moderate shear stress group and high shear stress group. Cultured endothelial cells of human cerebral arteriovenous malformation were put in a parallel plate flow chamber. In addition, cells in the low, moderate and high shear stress groups were stressed by low, moderate and high shear stress for 8 hours, respectively. However, shear stress in the control group was 0 Pa. Flow cytometry was used to measure proliferation index, and the expression of c-myc protein and c-myc mRNA were determined by immnocytochemistry and RT-PCR analysis respectively. MAIN OUTCOME MEASURES： Expressions of c-myc protein and c-myc mRNA and proliferation index in endothelial calls under various degrees of shear stress. RESULTS： ① Proliferation index： Proliferation index was higher in the moderate and high shear stress groups than that in the control group （P 〈 0.05, 0.01 ）. ②Expression of c-myc protein： Immunepositive expression of c-myc protein was gradually increased with the increase of shear stress and there were significant differences in the three shear stress groups as compared with control group （P 〈 0.05-0.01 ）. ③Expression of c-myc mRNA： Proliferation index of endothelial calls was higher in the low and moderate shear stress groups than that in the control group （P 〈 0.05）. CONCLUSION ： Flow shear stress can induce expression of c-myc and activate expression of c-myc gene based on gene transcription so as to promote the proliferation of vascular endothelial calls in human cerebral arteriovenous malformation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第36期7278-7281,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省博士科研启动基金资助(20061031)~~
