论文部分内容阅读
背景:观察缺氧状态下脑血管内皮细胞的血管内皮细胞生长因子表达及细胞超微结构变化,可为从细胞和分子水平认识血管生成及其相关的细胞因子参与缺血性脑血管疾病的病变过程。目的:构建分离微血管段体外培养人脑微血管内皮细胞的方法,并观察血管内皮细胞生长因子基因表达与细胞超微结构变化。设计:随机对照的技术方法研究。单位:一所军区总医院的神经外科,一所大学医院的神经外科。对象:2002年沈阳军区总医院神经外科脑动静脉畸形患者18例(SpetzlerⅡ～Ⅲ级),全部病例术前均经全脑血管造影证实。取材于手术中切除的完整脑动静脉畸形新鲜标本周围粘连的新鲜脑组织,采用匀浆、过滤和酶消化技术分离微血管内皮细胞。将在培养瓶内生长良好的细胞分成缺氧2,4,8h组和对照组4组,每4瓶为1组。方法:缺氧条件模拟:体积分数0.95N2,体积分数0.05CO2。免疫组化方法检测细胞中第八因子相关抗原(FⅧ-RA)表达。选用RT-PCR技术观察每组内皮细胞血管内皮细胞生长因子mRNA表达,同时以ELISA方法检测每组细胞上清液中血管内皮细胞生长因子蛋白含量,透射电镜观察细胞超微结构的变化。主要观察指标:对照组和各缺氧组血管内皮细胞中血管内皮细胞生长因子mRNA表达和细胞上清液中血管内皮细胞生长因子蛋白含量,以及细胞超微结构的变化。结果:相差显微镜下,培养的活细胞具有单层“卵石样”排列的典型特征,90%以上的细胞为FⅧ-RA阳性染色。缺氧4h细胞血管内皮细胞生长因子mRNA和蛋白表达为0.98±0.19,(180.77±20.15)ng/L,较对照组显著升高[0,(26.20±6.33)ng/L,P<0.01],8h后表达下调[0.35±0.07,(31.68±8.34)ng/L],并出现线粒体肿胀、内质网扩张及溶酶体多囊泡形成。结论:采用分离微血管段方法培养人脑血管内皮细胞,操作简便易行,细胞纯度可靠。缺血缺氧早期血管内皮细胞生长因子表达不足以维持细胞超微结构完整,随缺氧时间延长细胞可发生损伤性变化。
BACKGROUND: To observe the expression of vascular endothelial cell growth factor (VEGF) and the ultrastructure of cells in hypoxic condition of cerebrovascular endothelial cells can be used to recognize angiogenesis and its related cytokines involved in the pathogenesis of ischemic cerebrovascular diseases at the cellular and molecular level process. OBJECTIVE: To construct a method of culturing human brain microvascular endothelial cells in vitro and observe the changes of vascular endothelial growth factor (VEGF) gene expression and cell ultrastructure. Design: A Randomized Controlled Technical Methodology Study. Unit: Neurosurgery at a General Hospital of the Military Region and neurosurgery at a university hospital. PARTICIPANTS: Eighteen patients (Spetzler Ⅱ ~ Ⅲ) with neurosurgery cerebral arteriovenous malformations in Shenyang Military Region General Hospital in 2002 were confirmed by whole cerebral angiography before operation. Fresh brain tissue from the fresh specimens of intact cerebral arteriovenous malformations excised from the surgeries was isolated and homogenized, filtered and enzymatically digested to isolate microvascular endothelial cells. The well-growing cells in the culture flask were divided into hypoxia group 2,4,8h and control group 4, 4 bottles for each group. Methods: hypoxia condition simulation: volume fraction of 0.95N2, volume fraction of 0.05CO2. Immunohistochemistry was used to detect the expression of FⅧ-RA in the cells. RT-PCR was used to observe the mRNA expression of VEGF in each group. At the same time, the content of vascular endothelial growth factor in the supernatant of each group was measured by ELISA. The ultrastructure of the cells was observed by transmission electron microscope. MAIN OUTCOME MEASURES: The mRNA expression of vascular endothelial growth factor and the content of vascular endothelial growth factor in the supernatant of the control group and each hypoxia group, and the ultrastructure of the cells. Results: Under the phase-contrast microscope, the living cells cultured had the characteristic of monolayer “pebble-like” arrangement. More than 90% of the cells were FⅧ-RA positive staining. Compared with the control group, the mRNA and protein expression of VEGF in the 4 h hypoxia group was significantly higher than that in the control group [(0.20 ± 6.33) ng / L, 0.98 ± 0.19, (180.77 ± 20.15) ng / L, P <0.01] After 8h, the expression was down-regulated [0.35 ± 0.07, (31.68 ± 8.34) ng / L], with mitochondria swelling, endoplasmic reticulum dilation and lysosomal vesicle formation. Conclusion: The culture of human vascular endothelial cells by microvascular isolation is simple and easy to operate with reliable cell purity. In the early stage of hypoxia and hypoxia, the expression of vascular endothelial growth factor is not enough to maintain the integrity of the ultrastructure of cells, and the damage of cells may occur with the prolongation of hypoxia.