【关键词】 STZ大鼠；,糖尿病视网膜病变；,缺氧诱导因子；,血管内皮细胞生长因子
Expressions of hypoxiainducible factor1α and vascular endothelial growth factor in retina of streptozotocininduced diabetic rats
【Abstract】 AIM: To study the expressions of hypoxiainducible factor1α (HIF1α) and vascular endothelial growth factor (VEGF) in retina of streptozotocininduced diabetic rats. METHODS: Sixty Wister rats were divided into testing group (group T, 45 cases) and control group (group C, 15 cases). Streptozotocininduced diabetic animal models were made in group T, and then the rats were divided into 3 groups ( T1, T3, T5) according to the time (1, 3, 5 months) after modeling. The rats in group C were also divided into 3 groups (C1, C3, C5). The expression of VEGF was assessed by immunohistochemical method and the expression of HIF1α was assessed by Western Blotting. All the results were quantitatively analyzed by transformed into average optical density. RESULTS: 86.67%-93.33% of all the animal models were successful. Results of VEGF: There were no significant difference in the 3 control groups, as well as between group T1 and control groups (P>0.05); But the significant difference was found between group T3, T5 (t=6.12, P<0.01) and control groups (t=8.63, P<0.01) as well as between group T3 and T5 (t=3.45, P<0.05). Results of HIF1α: There was no significant difference in the 3 control groups and in the 3 testing groups (P>0.05). But the significant differences were found between group T1, T3, T5 and the 3 control groups (t=4.35, 5.01, 5.34, P<0.01). CONCLUSION: We observed the increased expression of VEGF and HIF1α in retina of streptozotocininduced diabetic rats, and the latter occurred earlier than the former. It suggests that the increased expression of HIF1α might be the initial factor for expression of VEGF during diabetic retinopathy.
【Keywords】 streptozotocininduced diabetic rats; diabetic retinopathy; hypoxiainducible factor; vascular endothelial growth factor
【摘要】 目的：探讨链脲佐菌素（STZ）糖尿病大鼠视网膜组织中缺氧诱导因子1α（HIF1α）和血管内皮生长因子（VEGF）的表达规律. 方法：Wister大鼠60只，分为实验组(T组,45只）和对照组(C组,15只）. 实验组建立STZ诱导的糖尿病大鼠模型，并随机分为T1，T3，T5三个组，每组15只，分别代表糖尿病模型1， 3， 5 mo;对照组同样随机分为C1，C3，C5三个组，每组5只. 分别采用免疫组织化学法检测视网膜VEGF表达、免疫印迹法检测视网膜HIF1α表达，结果以平均吸光度值进行定量比较. 结果：糖尿病模型成功率86.67％~93.33%. VEGF检测结果：对照组各组间差别无统计学意义（P＞0.05）；T1与对照组间差别无统计学意义（P＞0.05），但T3，T5与对照组间差别均有统计学意义（t值分别为6.12，8.67，P<0.01），并且T3，T5两组间差别也有统计学意义（t=3.45，P<0.05）. HIF1α检测结果：对照组各组间差别无统计学意义（P＞0.05）；T1，T3，T5分别与对照组相比，其差异均有统计学意义（t值分别为4.35，5.01，5.34，P<0.01），但实验组各组间差异无统计学意义（P＞0.05）. 结论：STZ糖尿病大鼠视网膜内可观察到VEGF和HIF1α的高表达，后者早于前者，提示在视网膜病变时HIF1α的表达可能是VEGF表达的始动因素.
【关键词】 STZ大鼠；糖尿病视网膜病变；缺氧诱导因子；血管内皮细胞生长因子
0 引言
糖尿病目前已成为严重危害公共健康的问题，可引起全身多数器官并发症，而其中微血管并发症之一的视网膜病变（diabetic retinopathy，DR），也日益成为致盲的主要原因. 有关DR的病理生理过程已有大量实验研究，并已发现血管内皮生长因子（vascular endothelial growth factor，VEGF）是引起DR血管渗漏、新生血管形成的主要细胞因素之一［1］. 然而， 在糖尿病患者眼部微环境下引起VEGF等细胞因子大量分泌的确切机制还不甚明了. 本实验我们从缺氧诱导因子1α（hypoxiainducible factor1α， HIF1α）这一途径， 探讨糖尿病条件下视网膜VEGF表达增加的机制.
1 材料和方法
1.1材料
选取封闭群雄性Wistar大鼠60只，体质量190～210 g，适应性饲养3 d，分为实验组（T组，45只）和对照组（C组，15只）. 将T组再随机分为T1，T3，T5三个组，每组15只，分别代表糖尿病模型1，3，5 mo；C组同样随机分为C1，C3，C5三个组，每组5只. 所有动物均在相同条件下饲养. 链脲佐菌素（streptoztocin, STZ)，美国Sigma公司产品；一抗为兔抗鼠VEGF mAb（1∶100），二抗为生物素化羊抗兔IgG，所有抗体和SABC试剂盒，DAB显色剂均购于美国DAKO公司;一抗（1∶200兔抗HIF1α多克隆抗体)，美国Sigma公司.
1.2方法
1.2.1糖尿病大鼠模型的建立［2］
取STZ溶于0.05 mol/L柠檬酸缓冲液(pH 4.5)，配置成浓度1.25%的溶液，按60 mg/kg对T组大鼠行一次性腹腔内注射，C组大鼠腹腔注射等量柠檬酸缓冲液. 药物注射后24～48 h检测血糖和尿糖，血糖浓度高于16.5 mmol/L，尿糖达～，尿量、饮水明显增多即为模型建立成功；低于上述标准则为造模失败. 实验组T1，T3，T5及对照组C1，C3，C5分别在造模成功后1，3，5 mo处死，取标本进行下列实验.
1.2.2免疫组织化学法（SABC）检测
视网膜VEGF的表达实验动物经10 g/L戊巴比妥钠（40 mL/kg）腹腔麻醉，用0.1 mo/L含有40 g/L多聚甲醛的PBS液灌流固定，摘除眼球，石蜡包埋后行16 μm连续切片，SABC法行免疫组织化学染色检查，其中对照组一抗以PBS液代替，操作步骤严格按照试剂盒说明书进行. 阳性结果为胞浆内出现棕褐色颗粒. 染色结果用LuzexF实时图像分析系统进行分析，计算平均A值.
1.2.3免疫印迹法（Western Blot）检测
视网膜HIF1α的表达实验动物同前麻醉，固定后，摘除眼球去除眼前段成眼杯，仔细分离出视网膜组织，剪碎后置于裂解液中机械匀浆，4℃，17 000 g离心30 min，取上清液以紫外分光光度计定量，等量蛋白上样进行75 g/L SDSPAGE电泳，电泳后湿转至硝酸纤维素膜上. 37℃孵育2 h，洗膜，加1∶1000的辣根过氧化物酶标记的二抗，37℃孵育1 h，洗膜后加入化学发光液反应1 min，曝光显影后冲洗胶片. 显色结果同样用图像分析系统计算平均A值.
统计学处理： 用SPSS 11.0统计软件对各实验数据进行分析处理，结果以x±s表示，采用Fisher确切概率计算法、多组间比较方差分析、组间比较t检验等进行差异显著性分析.
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