下肢缺血大鼠模型血管内皮细胞生长因子变化

Variational trend of vascular endothelial growth factor in rat models of lower limb ischemia

背景:组织缺血初期,机体通过代偿性调节与修复,实现血管的新生,使组织供血得以维持,在此代偿过程中,多种细胞因子,特别是血管内皮细胞生长因子发挥了关键的作用。目的:观察下肢缺血大鼠模型造模后不同时间点缺血组织及血清中血管内皮细胞生长因子水平变化特点及规律。设计、时间及地点:随机对照动物实验,于2007-04/11在东直门医院重点学科实验室和北京中医药大学分子生物实验室完成。材料:将4周龄雄性SD大鼠42只按照随机数字表分为6组:对照组、造模后4h、3d、1周、2周及4周组。方法:将模型组大鼠用100g/L水合氯醛麻醉后行左侧股总动脉结扎离断术制作下肢缺血模型,于造模成功后4h、3d、1周、2周及4周抽取大鼠腹主动脉血5mL,3000r/min离心10min后,取上清;同时分离大鼠左小腿腓肠肌组织。对照组大鼠麻醉后直接取材。主要观察指标:利用Western Blotting法检测缺血组织血管内皮细胞生长因子蛋白表达量、ELASA法检测血清中血管内皮细胞生长因子水平。结果:缺血组织血管内皮细胞生长因子在缺血后4h即开始增多,3d时达到高峰,3d后血管内皮细胞生长因子表达量逐渐减少,至缺血2周时血管内皮细胞生长因子表达量最少,2周后缓慢回升,至缺血4周时血管内皮细胞生长因子表达量接近正常组织。缺血后血清血管内皮细胞生长因子水平立即下降,缺血后即刻至缺血4h下降幅度最大,缺血4h～1周下降幅度减少;缺血1～4周血清血管内皮细胞生长因子少量增加,至缺血4周时仍显著低于正常组(P<0.01)。血清血管内皮细胞生长因子含量变化趋势表现为至缺血4h迅速下降,缺血1周降至最低点,1周后缓慢升高的趋势。结论:大鼠后肢缺血后,缺血组织中血管内皮细胞生长因子水平呈现先升高-再下降-再上升的趋势,血清中血管内皮细胞生长因子水平呈现先下降-再上升趋势,即呈现血清水平低,局部组织水平高的特点。

BACKGROUND： In the initial stage of ischemia, organisms complete angiogenesis and maintain the blood supply of organization through compensatory regulation and repair, in which process a variety of cytokines, especially vascular endothelial growth factor （VEGF）, have played a key role. OBJECTIVE： To observe the characteristics and rules of VEGF level changes in both ischemic tissues and blood serums at different time points after establishing rat models of lower limb ischemia. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at the Loboratory for Key Subjects in Dongzhimen Hospital and the Laboratory of Molecular Biology in Beijing University of Chinese Medicine from April to November in 2007. MATERIALS： A total of 42 male SD rats of 4 weeks were divided by random digits table into 6 groups, namely a control group and a model group which was subdivided into 5 groups at the time points of hour 4, day 3, weeks 1, 2 and 4 post modeling respectivly. METHODS： Rat models of lower limb ischemia were established in the model group by performing ligation and mutilation operation to left femoral arteries of rats that were anesthetized with 100 g/L chloral hydrate. At the time points of hour 4, day 3, weeks 1, 2 and 4 following modeling respectively, rats were selected to extract their abdominal aorta blood samples whose supernatant was then obtained through 10 minutes of 3 000 r/min centrifugalization. Gastrocnemius tissues in left legs of rats were isolated at the same time. Samples in control group were obtained directly from anesthetized rats. MAIN OUTCOME MEASURES： Western blotting method was used for detecting protein expression of VEGF in ischemic tissues and ELASA method for VEGF level in serum. RESULTS： The protein expression of VEGF in ischemic tissues began to increase immediately at hour 4 following ischemia and reached a peak at day 3, after which it reduced gradually till week 2 when it reached its minimum. Then it began to increase again and reached the level close to that of normal tissues by the end of week 4 following ischemia. As for the VEGF level in serum, it decreased immediately after ischemia, with the maximum decrease amplitude between immediate and hour 4 following ischemia, a smaller one between hour 4 and week 1 ; From week 1 following ischemia on, it began to increase but was still lower than the normal level by the end of week 4 （P 〈 0.01 ）. The VEGF level in serum changed with the tendency of immediate decrease from hour 4, minimum at week 1 and graduate increase after week 1 following ischemia. CONCLUSION： After ischemia in lower limb, VEGF level in ischemic tissues changes in the direction of increase-decrease-increase; VEGF level in serum changed in the direction of decrease-increase. In another words, VEGF levels after ischemia shows the characteristics of being low in serum and high in local ischemic tissues.