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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 some coding endothelial cells associated with angiogenesis cytokine gene, shear stress of blood is an important biological role in the regulation of vascular structures and functions. Objective: To observe the effects of fluid laminar shear stress on the proliferation of vascular endothelial cells and the expression of proto-oncogene c-myc in human cerebral arteriovenous malformations. Design: a randomized controlled technical method. Unit: PLA Shenyang Military Region General Hospital of Neurosurgery. PARTICIPANTS: Experiments were performed at the PLA Institute of Neuromedicine, Shenyang Military Region General Hospital, PLA from November 2006 to February 2007. 2006 people choose the PLA Shenyang Military General Hospital of Neurosurgery SpetzlerⅡ-Ⅲ grade 20 cases of cerebral arteriovenous malformation patients vein resection of cerebral arteriovenous malformation fresh specimens, before surgery in all cases were confirmed by cerebral angiography. M199 medium (Gi1bco BRL), high-quality fetal calf serum (HyClone), endothelial cell growth supplements (ECGS; U.S. Sigma), CO2 incubator (U.S. Forma Scientific), cell cycle analysis kit (BD Co.), flow cytometry (FACS Calibur, BD), mouse anti-human c-myc monoclonal antibody (Santa Cruz, USA) and RT-PCR kit (Promega). Methods: The vascular endothelial cells of human cerebral arteriovenous malformations were cultured by tissue adherent method. The cells were divided into 4 groups according to the stress of shear stress: control group, low-cut group, middle-cut group and high-cut group. The cultured human AVM monolayers were placed in the plate flow system. The cells in low-cut group, middle-cut group and high-cut group were exposed to low, medium and high shear stress for 8 h, respectively. The shear stress in the control group was 0 Pa. Cell proliferation index was determined by flow cytometry. Detection of c-myc protein expression. Detection of c-mycmRNA expression. MAIN OUTCOME MEASURES: Expression of c-myc protein and mRNA in endothelial cells and cell proliferation index under different shear stress. Results: ① Proliferation index: The proliferation index of endothelial cell in middle and high-cut group was higher than that in control group (P <0.05, 0.01). ② The expression of c-myc protein: The positive expression of c-myc protein increased with the increase of shear stress. There was significant difference between each stress group and the control group (P <0.05-0.01). ③ The expression of c-myc mRNA: The proliferation index of endothelial cells in low-cut group and middle-cut group was higher than that in control group (P <0.05). Conclusion: Laminar shear stress can induce the expression of c-myc at gene transcription level and promote the proliferation of human cerebral arteriovenous malformation vascular endothelial cells possibly by activating c-myc gene expression.