CAJ | 학술논문

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Spetzler Ⅱ-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8 h,对照组切应力为0 Pa.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-myc mRNA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. 揹景:切應力可以直接介導內皮細胞錶達一些編碼與血管生成相關的細胞因子基因,血液的流體切應力對調節血管結構和功能具有重要的生物學作用.目的:觀察流體層流切應力對人腦動靜脈畸形血管內皮細胞增殖與原癌基因c-myc錶達的影響.設計:隨機對照的技術方法.單位:解放軍瀋暘軍區總醫院神經外科.對象:實驗于2006-11/2007-02在解放軍瀋暘軍區總醫院全軍神經醫學研究所完成.選用2006解放軍瀋暘軍區總醫院神經外科Spetzler Ⅱ-Ⅲ級20例腦動靜脈畸形患者手術切除的人腦動靜脈畸形新鮮標本,全部病例術前均經全腦血管造影證實.M199培養液(Gi1bco BRL),優質胎牛血清(HyClone),內皮細胞生長添加劑(ECGS;美國Sigma),CO2培養箱(美國 Forma Scientific),細胞週期分析試劑盒(BD公司),流式細胞儀(FACS Calibur,BD公司),鼠抗人c-myc單剋隆抗體(美國Santa Cruz公司),RT-PCR試劑盒(Promega).方法:採用組織塊貼壁法培養人腦動靜脈畸形血管內皮細胞,按所受切應力大小將細胞分為4組:對照組、低切組、中切組和高切組.將培養的人腦動靜脈畸形內皮細胞單層置于平闆流動繫統內,低切組、中切組和高切組細胞分彆經低、中、高切應力作用8 h,對照組切應力為0 Pa.應用流式細胞術測定細胞增殖指數.檢測c-myc蛋白錶達.檢測c-mycmRNA錶達.主要觀察指標:不同切應力作用下內皮細胞c-myc蛋白及mRNA錶達和細胞增殖指數.結果:①細胞增殖指數:中切組和高切組內皮細胞增殖指數高于對照組(P＜0.05,0.01).②c-myc蛋白錶達:c-myc免疫暘性錶達隨切應力增高而遞增,各切應力組與對照組比較差異均有統計學意義(P＜0.05～0.01).③c-myc mRNA錶達:低切組和中切組內皮細胞增殖指數高于對照組(P＜0.05).結論:流體層流切應力能夠在基因轉錄水平誘導c-myc錶達,併可能通過激活c-myc基因錶達而促進人腦動靜脈畸形血管內皮細胞增殖.
배경:절응력가이직접개도내피세포표체일사편마여혈관생성상관적세포인자기인,혈액적류체절응력대조절혈관결구화공능구유중요적생물학작용.목적:관찰류체층류절응력대인뇌동정맥기형혈관내피세포증식여원암기인c-myc표체적영향.설계:수궤대조적기술방법.단위:해방군침양군구총의원신경외과.대상:실험우2006-11/2007-02재해방군침양군구총의원전군신경의학연구소완성.선용2006해방군침양군구총의원신경외과Spetzler Ⅱ-Ⅲ급20례뇌동정맥기형환자수술절제적인뇌동정맥기형신선표본,전부병례술전균경전뇌혈관조영증실.M199배양액(Gi1bco BRL),우질태우혈청(HyClone),내피세포생장첨가제(ECGS;미국Sigma),CO2배양상(미국 Forma Scientific),세포주기분석시제합(BD공사),류식세포의(FACS Calibur,BD공사),서항인c-myc단극륭항체(미국Santa Cruz공사),RT-PCR시제합(Promega).방법:채용조직괴첩벽법배양인뇌동정맥기형혈관내피세포,안소수절응력대소장세포분위4조:대조조、저절조、중절조화고절조.장배양적인뇌동정맥기형내피세포단층치우평판류동계통내,저절조、중절조화고절조세포분별경저、중、고절응력작용8 h,대조조절응력위0 Pa.응용류식세포술측정세포증식지수.검측c-myc단백표체.검측c-mycmRNA표체.주요관찰지표:불동절응력작용하내피세포c-myc단백급mRNA표체화세포증식지수.결과:①세포증식지수:중절조화고절조내피세포증식지수고우대조조(P＜0.05,0.01).②c-myc단백표체:c-myc면역양성표체수절응력증고이체증,각절응력조여대조조비교차이균유통계학의의(P＜0.05～0.01).③c-myc mRNA표체:저절조화중절조내피세포증식지수고우대조조(P＜0.05).결론:류체층류절응력능구재기인전록수평유도c-myc표체,병가능통과격활c-myc기인표체이촉진인뇌동정맥기형혈관내피세포증식.
BACKGROUND:Shear stress can directly mediate the expression of endothelial cells, 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.OBJECTIVE: To observe the effects of laminar flow shear stress on the proliferation of vascular endothelial cells and the expression of protooncogene c-myc 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 carried 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 (HyClone), 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 cells 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 cells 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). ② Expression of c-myc protein: Immuneposjtjve 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). ③ Expression of c-myc mRNA: Proliferation index of endothelial cells 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 cells in human cerebral arteriovenous malformation