银杏叶提取物对豚鼠视神经横断伤后视网膜神经节细胞结构及功能的

1、银杏叶提取物对豚鼠视神经横断伤后视网膜神经节细胞结构及功能的保护作用         09-12-13 16:04:00     编辑：studa20                作者：解正高, 吴星伟, 庄朝荣, 陈放, 王正, 王雅坤, 华欣【摘要】  目的: 探讨腹腔注射银杏叶提取物（Ginkgo biboba

2、 extract，EGb 761）对豚鼠视神经横断伤后视网膜神经节细胞（retinal ganglion cell, RGC）形态及功能的保护作用。方法: 75只白化豚鼠随机等分为正常对照组、假手术组、模型组、生理盐水组和EGb 761组。分离暴露豚鼠的右眼视神经并于球后1.0 mm处进行横断造模，正常对照组不作任何处理，假手术组仅分离暴露视神经。生理盐水组和EGb 761组分别于实验开始前1周每日腹腔注射1次相应体积生理盐水和EGb 761（100 mg/kg），术后继续给药4周。术后第4天，各组随机处死3只豚鼠，采用脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxyn

3、ucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick endlabeling, TUNEL)法检测RGC凋亡；分别于术后第14和28天进行图形视网膜电图（pattern electoretinogram, PERG）检查；摘取眼球作组织病理学检查并计数视网膜垂直经线RGC数目。结果: 术后第4天，正常对照组、假手术组和EGb 761组均未见TUNEL阳性RGC，模型组和生理盐水组均见有TUNEL阳性RGC。术后第14和28天，模型组和生理盐水组RGC数目均少于正常对照组和假手术组（P<0.05），EGb 7

4、61组少于正常对照组和假手术组（P<0.05），但显著多于模型组和生理盐水组（P<0.05）；术后第14和28天，EGb 761组PERG的N95振幅较模型组和生理盐水组高（P<0.05），与模型组和假手术组相近（P>0.05）。RGC数目与N95振幅呈正相关（r=0.859, P=0.001 5）。结论: 腹腔注射银杏叶提取物EGb 761能抑制豚鼠视神经横断后的RGC凋亡，对RGC的结构和功能具有保护作用。 【关键词】  银杏叶提取物; 视神经损伤; 视网膜神经节细胞; 图形视网膜电图; 神经保护; 豚鼠Objective: To investigate

5、the effects of Ginkgo biloba extract (EGb 761) on the morphology and function of retinal ganglion cells (RGC) in guinea pigs with optic nerve transection.Methods: Seventyfive albino guinea pigs were randomly divided into five groups: normal control group, shamoperated group, untreated group, normal

6、saline group and EGb 761 group. No operation was performed in the normal control group. Optic nerve was merely exposed in the shamoperated group, but transected at 1.0 mm from posterior pole of the eye ball in the untreated, normal saline and EGb 761 groups. Guinea pigs in the EGb 761 group or the n

7、ormal saline group received daily intraperitoneal injection of EGb 761 (100 mg/kg) or corresponding volume of normal saline from 7 days before experiment to 28 days after experiment. Three guinea pigs in each group were sacrificed for apoptosis assay (TUNEL method) of RGC. Pattern electoretinograms

8、(PERGs) were recorded 14 and 28 days after transection, respectively. At the end of the examination, six guinea pigs were killed for histological examination and RGC count.Results: No TUNELpositive cells were observed in the normal control, shamoperated and EGb 761 groups, but there were TUNELpositi

9、ve cells in the untreated group and the normal saline group. The numbers of RGCs in the untreated and normal saline groups were less than those in the normal control and shamoperated groups at 14 days or 28 days (P<0.05). Although the number of RGCs in the EGb 761 group was less than those in the

10、 normal control and shamoperated groups (P<0.05), it was more than those in the untreated and normal saline groups (P<0.05). N95 amplitude in EGb 761 group was higher than those in the untreated and normal saline groups (P<0.05) and close to those in the normal control and shamoperated grou

11、ps (P>0.05) at 14 days or 28 days. The number of RGCs was positive correlated to N95 amplitude (r=0.859, P=0.001 5).Conclusion: EGb 761 can inhibit the apoptosis of RGCs in guinea pigs after optic nerve transection, thus protect the morphology and function of RGCs.Keywords: Ginkgo biloba extract;

12、 optic nerve lesion; retinal ganglion cell; pattern electroretinogram; neuroprotective effect; guinea pigs青光眼是一种威胁视神经视觉功能，主要与眼压升高有关的眼病，也是目前我国第二大致盲眼病。虽然青光眼的具体发病机制尚不清楚，但目前一致认为视网膜神经节细胞（retinal ganglion cell, RGC）凋亡为青光眼神经损害的中心环节。研究证明银杏叶提取物（Ginkgo biloba extract，EGb 761）具有抗氧化1、抗缺血2、保护线粒体3和抑制一氧化氮合酶活性4等广泛的

13、生物学效应。因此，我们采用横断豚鼠球后视神经的方法建立RGC损伤模型观察EGb 761对RGC结构的保护作用，并采用图形视网膜电图（pattern electroretinogram, PERG）评价EGb 761对RGC功能的保护作用。1  材料和方法1.1  实验材料1.2  实验方法1.3  统计学方法  所有数据采用x±s表示。采用SAS 6.12统计软件包进行单因素方差分析，如差异有统计学意义，则进行各组均数间两两比较（StudentNewmanKeuls test, SNK）；RGC数目和P50波振幅相关性检测采用直线相关

14、分析。检验水准=0.05。2  结果2.1  EGb 761对RGC凋亡的影响  凋亡细胞的细胞核在3通道激发光下呈绿色荧光着染，所有细胞核在5通道激发光下呈红色荧光着染，在8通道激发光下凋亡细胞的细胞核呈黄绿色或黄色荧光着染。正常对照组和假手术组视网膜神经节细胞层均未见黄色荧光着染的RGC。模型组和生理盐水组均见有较多强荧光着染的RGC。EGb 761组未见荧光着染的RGC。见图1。2.2  光学显微镜观察  豚鼠视网膜可见清晰的10层组织学结构，但全层无血管样结构。正常对照组神经纤维层较厚，RGC呈立方形，排列紧密，胞浆丰富，核饱满，染色较

15、均匀，而胶质细胞胞体较小，核深染。假手术组除RGC略为稀疏外，与正常对照组无明显差异。视神经横断后28 d，模型组和生理盐水组RGC的数目明显减少，神经纤维层明显变薄且RGC稀少，胞体变小。EGb 761组RGC胞体与正常大小无明显区别，但数目略少。见图2。2.3  EGb 761对RGC存活指数的影响  正常豚鼠视网膜垂直经线上RGC的数目为（318±13）个。视神经横断后14 d时，模型组与生理盐水组RGC存活指数显著低于正常对照组和假手术组（P<0.05）；EGb 761组虽比正常对照组和假手术组低（P<0.05），但显著高于模型组和生理盐水组（

16、P<0.05）。视神经横断后28 d时，模型组与生理盐水组RGC存活指数进一步降低，显著低于正常对照组和假手术组（P<0.05）；EGb 761组RGC存活指数也有所降低，虽比正常对照组和假手术组低（P<0.05），但显著高于模型组和生理盐水组（P<0.05）。见图3。图1  TUNEL检测EGb 761对RGC凋亡的影响（荧光显微镜，×400）（略）Figure 1  Effects of EGb 761 on RGC apoptosis checked by TUNEL (Fluorescein microscopy, ×400)GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; TUNEL: DeadEnd fluorometric terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick endlabeling; PI: Propidium iodide. A: Normal control group, no TUNEL positive RGCs; B: Shamoperated