摘要
目的：在NMDA所致大鼠视网膜兴奋损伤模型中,检测谷氨酰胺诱导HSP70的表达及随时间的变化。
方法：健康清洁型Wistar大鼠70只随机分为三组,正常对照组10只,空白对照组10只、实验组即谷氨酰胺腹腔注射组50只。正常对照组不做任何处理,空白对照组和实验组中所有大鼠均随机取大鼠一只眼球玻璃体腔注射2μ l80mmol/lNMDA制作视网膜兴奋性损伤模型,实验组在大鼠视网膜损伤兴奋性损伤模型制作2天后分别腹腔注射谷氨酰胺(0.75g/Kg),并于注射后1h,4h,8h,12h,24h各随机处死10只大鼠,其中1h时同时将空白对照组及正常对照组所有大鼠处死。HE染色光镜下观察各组视网膜组织的形态学变化,免疫组织化学染色方法及Western Blot分析视网膜HSP70表达的变化。
结果：HE染色显示正常对照组大鼠视网膜组织结构层次清楚,染色均匀,细胞形态规则。空白对照组与实验组中不同时间段的大鼠视网膜均显示视网膜神经节细胞数目较正常大鼠减少,排列紊乱,神经纤维层与内核层可见萎缩,视网膜整体变薄。Western Blot分析结果显示,正常对照组和空白对照组仅有少量HSP70的表达。实验组注射1h后HSP70的表达开始增强,4h后表达明显增多,8h达到高峰并持续到12h,24h表达较前减少,但仍显著高于对照组。免疫组织化学染色显示诱导表达的HSP70在视网膜各层细胞均有显著表达,而正常对照组及空白对照组HSP70仅微量表达于外核层及光感受器内节。免疫组化染色阳性细胞光密度值测定结果与Westefn Blot结果相吻合。
结论：1.成功建立大鼠视网膜兴奋性损伤模型；
2.正常情况下和玻璃体腔注射NMDA2天后大鼠视网膜微量表达HSP70；
3.腹腔注射谷氨酰胺可以在大鼠视网膜兴奋性损伤中诱导HSP70的强烈表达。
PURPOSE. To observe the effects of glutamine (GLN) on the expression of HSP70in the rat's retina excitotoxicity injury induced by NMDA.
METHODS.A total of70rats were randomly divided into three groups, normal control group including10rats, blank control group including10rats, experimental group in which rats were treated with glutamine including50rats. The rats of normal control group received no treatment.2μ1of NMDA of concentration of80nmol/1was intravtreal injected into the one random eye of every animal of experimental group and blank control group.After two days, the rats in experimental group were intraperitoneal injected with glutamine (0.75g/kg) and10rats were sacrificed at lh,4h,8h,12h,24h randomly and respectively after glutamine administration. The rats in normal control group and blank control group were sacrificed at1h. The rats'retina were HE stained and observed under light microscope, and the expression level of HSP70were detected by immunohistochemistry and Western blot analysis.
RESULT. HE staining show the structure of retina of normal control group was histologically well-defined, uniform in staining, and with regular cell shape. In blank control group and experimental group, the retina ganglion cells (RGCs) with striking chromatin condensation were loosely arranged and fewer RGCs were found in the retina that became atrophic and thinner. The Western blot analysis showed there was slight expression of HSP70in normal control group and blank control group retinas. The expression of HSP70was observed increase at the lth hour after being injected with GLN, a strong increase was detected at4th hour, and reached the peak at the8th hour; until12th hour. There was a mild decrease of HSP70expression in the24th hour, but it was still stronger than the control retinas. The expression of HSP70was detected in cells of almost all layers of retinas by immunohistochemical staining after GLN administration. Compares with the experimental group, Hsp70immunostaining was only observed in the inner segment of photoreceptors, the outer limiting membrane layer and the outer nuclear layer (ONL) of the normal control and the blank control group rat retina. The result of the quantitative analysis of immunoreactive intensity of HSP70in retina was similar to the result of Western blot.
CONCLUSION
1.The rat's retina excitotoxicity injury model has been set up successfully.
2. There was slight HSP70expressed in normal retina.
3. HSP70can be induced by glutamine intraperitoneal injection in the retina excitotoxicity injury and the level of expression changed with time.
引文
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