【摘要】 背景:碱性成纤维细胞生长因子是一种具有广泛神经活性的多肽生长因子,能保护神经元,促进神经生长。有证据证实碱性成纤维细胞生长因子可治疗视网膜缺血再灌注损伤。目的:建立视网膜缺血再灌注损伤动物模型,分析碱性成纤维细胞生长因子对其视网膜细胞凋亡及调控基因蛋白表达的影响。设计:随机分组,验证性实验。单位:青岛大学医学院附属医院眼科。材料:实验于2002-04/2003-12在青岛大学医学院眼科病理研究室完成。选择健康Wistar大鼠28只,随机取4只作为正常对照组,其余24只大鼠的左眼作为生理盐水对照组,右眼作为碱性成纤维细胞生长因子组。方法:生理盐水对照组、碱性成纤维细胞生长因子组采用前房加压法制作大鼠视网膜缺血再灌注损伤模型。生理盐水对照组从玻璃体腔注入生理盐水12μL,碱性成纤维细胞生长因子组从玻璃体腔注入碱性成纤维细胞生长因子12μL,4只/次。正常对照组不给药。分别于缺血1h再灌注1,6,12,24,48,72h6个时间点采用原位缺口末端标记、免疫组织化学染色检测凋亡细胞的表达,计算凋亡指数。主要观察指标:①各组大鼠再灌注后不同时间点视网膜组织原位凋亡细胞检测结果。②各组大鼠再灌注后不同时间点视网膜组织中Fas、半胱氨酸天冬氨酸蛋白酶-2的表达。结果:①缺血再灌注大鼠不同灌注时间下视网膜组织凋亡指数的比较:正常对照组大鼠视网膜中未见凋亡细胞。与生理盐水对照组比较,碱性成纤维细胞生长因子组于缺血1h再灌注1,6,12,24,48,72h6个时间点凋亡指数均明显降低,且再灌注12,24,48h时差异显著(t=5.362～5.595,P<0.05)。②缺血再灌注大鼠不同灌注时间下Fas表达的变化:正常对照组大鼠视网膜中几乎无Fas表达。与生理盐水对照组比较,碱性成纤维细胞生长因子组于缺血1h再灌注1,6,12,24,48,72h6个时间点Fas表达均明显降低,且再灌注6,12,24h时差异显著(t=3.954～9.327,P<0.05)。③缺血再灌注大鼠不同灌注时间下半胱氨酸天冬氨酸蛋白酶-2表达的变化:正常对照组大鼠视网膜中半胱氨酸天冬氨酸蛋白酶-2蛋白不表达。与生理盐水对照组比较,碱性成纤维细胞生长因子组于缺血1h再灌注6,12,24,48,72h半胱氨酸天冬氨酸蛋白酶-2表达均明显降低(t=4.125～15.641,P<0.05)。结论:碱性成纤维细胞生长因子可显著抑制凋亡基因Fas及半胱氨酸天冬氨酸蛋白酶-2在视网膜缺血再灌注损伤中的表达,从而减少神经节细胞凋亡,对视网膜缺血再灌注损伤起到治疗作用。更多还原

【Abstract】 BACKGROUND Basic fibroblast growth factor bFGF a kind of polypeptide growth factor possessing multifunctional biological activities can protect neurons and promote the growth of nerves. It has been confirmed that bFGF has therapeutic effects on retina ischemia/reperfusion injury RIRI. OBJECTIVE To establish RIRI model and analyze the effects of bFGF on cellular apoptosis of retina and the expression of regulatory gene protein. DESIGN Randomized grouping and validating trial. SETTING Department of Ophthalmology the Affiliated Hospital of Medical College of Qingdao University. MATERIALS The experiment was conducted at the Research Laboratory of Pathology Department of Ophthalmology Medical College of Qingdao University from April 2002 to December 2003. Twenty-eight healthy Wistar rats were enrolled in this experiment. Four rats were randomly chosen for normal control group the left eyes of the other 24 rats were set as normal saline control group and the right eyes were set as bFGF group. METHODS Normal saline control group and bFGF group adopted the rat RIRI models established by transiently elevating intraocular pressure. Normal saline of 12 μL was injected into the vitreous cavity of the left eyes of the rats in normal control group.12 μL bFGF was injected into the vitreous cavity of the right eyes of the rats in bFGF group 4 rats once. No administration was given in normal control group. The expression of apoptotic cells was detected and apoptosis indexes were calculated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling TUNEL method and immunohistochemical staining method at the 1st 6th 12th 24th 48th and 72nd hours after reperfusion and ischemia for 1 hour. MAIN OUTCOME MEASURES ① The detection results of apoptotic cells in situ of retina tissues at different time points after reperfusion. ② The expression of Fas and caspases-2 in retina tissues at different time points after reperfusion. RESULTS ① Comparison of apoptosis indexes of retina tissues at different time points after ischemia reperfusion There were no apoptotic cells in the retina tissues of the rats in normal control group. As compared with those in normal saline control group apoptosis indexes in bFGF group were significantly decreased at ischemia 1 hour and reperfusion 1 6 12 24 48 and 72 hours especially at the 12th 24th and 48th hours after reperfusion t =5.362-5.595 P < 0.05. ② The change of Fas expression at different time points after ischemia reperfusion There was hardly any Fas expression in normal control group. As compared with that in normal saline control group Fas expression in bFGF group was significantly decreased at ischemia 1 hour and reperfusion 1 6 12 24 48 and 72 hours especially at the 6th 12th and 24th hours after reperfusion t=3.954-9.327 P < 0.05. ③ The changes of caspase-2 expression at different time points after ischemia reperfusion There was no caspase-2 expression in normal control group. Compared with that in normal saline control group the number of caspase-2 positive cells in bFGF group was significantly decreased at the 6th 12th 24th 48th and 72nd hours after ischemia for 1 hour and reperfusion t =4.125-15.641 P < 0.05. CONCLUSION bFGF can significantly inhibit the expression of apoptosis gene Fas and caspase-2 in the ischemia and reperfusion of retina thus reducing cellular apoptosis of ganglion cells and exerting therapeutic effects on the ischemia and reperfusion of retina.更多还原