VEGF及其受体在脊髓缺血再灌注损伤中的表达及Bosentan干预的研究

摘要

目的 现察内皮素受体阻断剂Bosentan对大鼠脊髓缺血再灌注损伤(SCIRI)后血管内皮因子(VEGF)及其两个受体表达的变化和意义.方法 成年SD大鼠120只建立SCIRI模型,随机分为正常组、假手术组、单纯缺血组、生理盐水对照组(NS)、Bosentan干预组(Bos),NS组和Bos组按照再灌注(IR)时间分为6、12、24h和3、5、7d组,测定血清VEGF含量,采用逆转录-Real-time PCR、免疫组织化学染色检测Bosentan干预后Flk及Flt的表达变化.结果 ①IR后各时间点血浆VEGF含量均显著升高(P＜0.05或P＜0.01),以24h最高(P＜0.01),同时间点Bosentan干预组较IR血浆VEGF含量均显著增高(P＜0.05).②Bosentan干预后各时间点VEGF、FLK、FLT的蛋白表达均较NS组显著增强(P＜0.05或P＜0.01),以24h最明显(P＜0.05或P＜0.01).VEGF不仅在脊髓前角神经元表达,在Bosentan干预12、24h和3d时胶质细胞中亦有表达.FLK、FLT的蛋白仅在神经元胞浆中表达.③Bosentan干预后各组VEGF mRNA、FLK mRNA(7d除外)、FLT mRNA(7d除外)含量均较IR生理盐水对照组显著增高(P＜0.05或P＜0.01),以24h最为显著(P＜0.05或P＜0.01).结论 缺血再灌注损伤本身可诱导VEGF及其受体的表达,内皮素受体阻断剂Bosentan可使VEGF及其受体的表达增加,改善脊髓病理形态,对SCIRI过程的改善可能有益.%Objective To study the expression and its significance of VEGF and its receptors after Bosentan on ischemia reperfusion injury of spinal cord. Methods 120 rats were randomly divided into normal group (N), sham group (Sham), ischemia group (I), ischemia-reperfusion injury group with the saline as the control group (NS) and the group of Bosentan (Bos). According to different reperfusion time, the NS and Bosentan groups were divided into 6h. 12h, 24h. 3d. 5d and 7d group. The serum VEGF content was determinated. The expression of VEGF and its receptor were study with retrovirus-Real-time PCR and immunohistochemistry. Results The plasma VEGF in NS group was significantly higher than that in the normal group and the sham group (P <0. 05). Compared with the I group, the levels of plasma VEGF were significantly increased in NS group(P<0. 05, P<0. 01). The VEGF levels in plasma of Bos group significantly increased (P<0. 05). VEGF mRNA levels increased gradually till 7d (P<0. 05; P<0. 01). IR group FLK mR-NA level was gradually rise till IR5d(P<0. 05) P<0. 01). FLT mRNA level was gradually rise till 3d. The lever of VEGF and its receptor mRNA content in Bos group significantly increased in each group at same time point (except outside 7d) (P<0. 05;P<0. 01). The expressions of VEGF, FLK and FLT were negative of both motor neurons and glial cells in normal group and the Sham group. VEGF, FLK, FLT (7d excluded) expressed more significantly than ischemia group (P<0. 05, P<0. 01). after the intervention of Bosentan. VEGF, FLK and FLT protein expressed more significantly than IR (P<0. 05, P<0. 01). Conclusion Ischctnia-reperfusion injury itself can induce the expression of VEGF and its receptor. Bosentan which is endothelin receptor blockcrs can increase the expression of VEGF and its receptor.