尿激酶对慢性青光眼大鼠视网膜神经节细胞的保护作用

发布时间：2018-01-16 09:25

本文关键词：尿激酶对慢性青光眼大鼠视网膜神经节细胞的保护作用　出处：《新乡医学院》2014年硕士论文　论文类型：学位论文

【摘要】：背景青光眼是一种常见的致盲性疾病,其发病率有相对增长趋势。公认的唯一有效的治疗方法是降低眼内压,但眼压正常后并不能阻止视神经损伤的进展。视神经损害的最终结果是视网膜神经节细胞(RGCs)的凋亡,而目前视神经节细胞保护药物尚无确切有效的依据而应用于临床。目的建立大鼠青光眼压模型,观察尿激酶对慢性青光眼大鼠视网膜神经节细胞和神经纤维层(NFL)毛细血管内皮细胞P-JNK蛋白表达的影响；探讨尿激酶对慢性青光眼视神经的保护机制,为临床治疗提供理论依据。方法将45只健康SD雄性大鼠,随机分组,分别为正常组、高眼压组和尿激酶组,每组15只,采用巩膜上静脉灼烧烙闭法建立慢性高眼压青光眼模型。高眼压组和治疗组分别建立青光眼慢性高眼压模型,正常对照组不作任何处理.用Tono-pen AVVI手持笔式眼压计测量大鼠眼压。在7-10天后眼压稳定,治疗组给予腹腔注射尿激酶,正常组和高眼压组给予等量的生理盐水。造模1月后取材(眼球),分别处理后,行视网膜常规HE染色、P-JNK免疫组化和免疫荧光检测及电镜照相。采用SPSS13.0统计软件处理数据。各组测量数据以均数士标准差(±s)表示,三组总体比较采用方差分析,其中两组间的比较采用LSD-t检验,以P=0.05为标准进行比较。结果 1眼压测量制模前,各组大鼠的平均眼压在统计上没有显著性差异(P0.05)。制模后,高眼压组、尿激酶组眼压显著升高,统计上存在显著性差异(P0.05),1个月内眼压无明显的波动。在整个过程中,高眼压组和尿激酶组分别与正常对照组相比有统计学的显著性差异(P0.05)。而高眼压组和尿激酶组相比,眼压无统计学显著性(P0.05)。 2HE染色在正常对照组,视网膜外核层、内核层和神经节细胞(RGCs)排列整齐,细胞核规则。在高眼压组,外核层、内核层和RGCs排列疏松,不规整,其RGCs数目明显减少。尿激酶组外核层和内核层细胞排列稍紊乱,但较高眼压组清晰,RGCs数目较正常组略微下降,三组RGCs数总体比较,差异有统计学意义(P0.05)。 3P-JNK免疫组化检测正常组视网膜NLF和内颗粒层细胞基本未发现P-JNK蛋白阳性表达,慢性高眼压组NLF中RGCs和血管内皮细胞及内核层细胞发现大量的P-JNK蛋白阳性表达,尿激酶治疗组中RGCs P-JNK蛋白阳性表达明显减少,血管内皮细胞和内颗粒层未发现P-JNK蛋白阳性表达。三组视网膜平均灰度值总体比较,差异有统计学意义(P0.05)。高眼压组P-JNK平均灰度值与正常组比较,差异有统计学意义(P0.05)；尿激酶治疗组与其高眼压组组比较,差异有统计学意(P0.05),尿激酶治疗组与正常组比较,差异有统计学意(P0.05)。 4P-JNK免疫荧光检测正常组视神经鞘膜外纤维细胞基本上未发现P-JNK蛋白阳性表达,慢性高眼压组有较少的P-JNK蛋白阳性表达,尿激酶治疗者P-JNK蛋白阳性表达较高眼压组略有增强。三组视网膜神经节细胞层P-JNK平均荧光强度总体比较,差异有统计学意义(P0.05)。高眼压组P-JNK平均灰度值与正常组比较,差异有统计学意义(P0.05)；尿激酶治疗组与其高眼压组组比较,差异有统计学意(P0.05),尿激酶治疗组与正常组比较,差异有统计学意(P0.05)。 5电镜结果 5.1RGCs的电镜观察正常组,RGCs核形态规则,核周间隙正常,其染色质分布均匀,没有发现边集现象。细胞器没有看见太明显的水肿现象。质膜完整。高眼压组,RGCs形态不规则,核周间隙明显增宽,其染色质分布不均匀,有边集现象。细胞器呈现高度的水肿现象伴不完整的质膜。用药组,RGCs核形态规则,核周间隙增基本正常,其染色质分布均匀,边集现象不明显,细胞器未见很明显的水肿现象,质膜基本完整。 5.2视网膜NLF毛细血管的电镜观察正常组,血管壁完整,细胞排列规整。高眼压组,血管壁细胞遭到严重破坏,基底膜破裂,缺乏一个完整的血管壁。治疗组的完全血管壁细胞、内皮细胞和周细胞轻度水肿,基底膜轻度增厚,整体形状接近正常组。结论 1尿激酶可能是通过改善视网膜的微循环和增强视神经轴突的修复作用,间接抑制RGCs的凋亡及改善或延迟视神经轴突变性。 2提示视网膜局部的溶栓治疗对青光眼视功能保护的重要性。
[Abstract]:Background glaucoma is a common disease causing blindness, its incidence has a relative growth trend. The only effective treatment is known to reduce intraocular pressure, but did not stop the progress of normal intraocular pressure after optic nerve injury. The final result of optic nerve damage of retinal ganglion cells (RGCs) apoptosis, and present the ganglion cells protection drugs is no effective basis for clinical application.
Objective to establish a rat model of glaucoma, intraocular pressure, urokinase on chronic glaucoma rat retinal ganglion cell and nerve fiber layer (NFL) on expression of P-JNK protein of capillary endothelial cells; the mechanism of the protective effect of urokinase on chronic glaucoma optic nerve, and provide a theoretical basis for clinical treatment.
Methods 45 healthy male SD rats were randomly divided into normal group, respectively, high intraocular pressure group and urokinase group, 15 rats in each group, using the sclera to establish the model of chronic ocular hypertension glaucoma. Glaucoma with closed vein burning chronic high intraocular pressure model were established with high intraocular pressure group and treatment group, normal control group without any treatment. Tono-pen AVVI handheld Pen tonometer intraocular pressure in rats. In 7-10 days after the intraocular pressure stable, the treatment group was given intraperitoneal injection of urokinase, normal saline group and hypertension group were given the same amount of modeling. After January (the eye), were respectively treated by retinal routine HE staining, P-JNK immunohistochemistry immunofluorescence and electron microscopy and radiography. Using SPSS13.0 statistical software to process data. The measurement data were expressed as mean + standard deviation (+ s) said that the three groups were analyzed using analysis of variance, LSD-t test was used to compare between the two groups in the P=0.05 is a standard comparison.
Result
1 intraocular pressure measurement
Die, there is no significant difference in the statistics on the average intraocular pressure of rats (P0.05). After the mold, high intraocular pressure group, urokinase group significantly increased intraocular pressure, there are statistically significant differences (P0.05), intraocular pressure within 1 months without obvious fluctuations. In the whole process, significant difference in height the intraocular pressure group and urokinase group respectively compared with normal control group is statistically significant (P0.05). The high intraocular pressure group and urokinase group compared to IOP was not statistically significant (P0.05).
2HE staining
In the normal control group, the retinal outer nuclear layer, inner nuclear layer and ganglion cells (RGCs) arranged in nuclear rules. In high intraocular pressure group, the outer nuclear layer, inner nuclear layer and RGCs arranged loosely and irregularly, the number of RGCs decreased significantly. The urokinase group outer nuclear layer and inner nuclear layer cells arranged a little disorder, but higher the intraocular pressure group is clear, the number of RGCs than the normal group decreased slightly, the number of RGCs was three overall comparison, the difference was statistically significant (P0.05).
Immunohistochemical detection of 3P-JNK
NLF in normal retina and inner granular layer cells were found in the expression of P-JNK protein, RGCs and vascular endothelial cells and the inner nuclear layer cells found positive expression of P-JNK protein of the liftreatment group NLF, RGCs P-JNK protein positive expression of urokinase in the treatment group were significantly reduced, vascular endothelial cells and inner granular layer was found in the positive expression of P-JNK protein. Generally the average gray value of retinal group three, the difference was statistically significant (P0.05). Compared with the normal group, the average gray value of P-JNK high intraocular pressure group, the difference was statistically significant (P0.05); compared with urokinase treatment group high intraocular pressure group, there was statistically significant difference (P0.05), urokinase treatment group compared with the normal group, there was statistically significant difference (P0.05).
4P-JNK immunofluorescence detection
Normal optic nerve sheath fiber cells and expression of P-JNK protein was not found basically, the liftreatment group had less P-JNK protein expression, P-JNK protein expression of urokinase in the treatment of those high intraocular pressure group is slightly enhanced. Three groups of retinal ganglion cell layer P-JNK average fluorescence intensity overall comparison, the difference was statistically significant (P0.05). Compared with the normal group, the average gray value of P-JNK high intraocular pressure group, the difference was statistically significant (P0.05); compared with urokinase treatment group high intraocular pressure group, there was statistically significant difference (P0.05), urokinase treatment group compared with the normal group, there was statistically significant difference (P0.05).
5 electron microscope results
Electron microscopic observation of 5.1RGCs
The normal group, RGCs nuclear shape, nuclear week gap is normal, the chromatin distribution, no set of edges found. The organelles did not see obvious edema. Membrane integrity. High intraocular pressure group, RGCs irregular shape, nuclear week gap widened significantly, the chromatin of uneven distribution, edge set. Cell organelle showed edema with highly incomplete nuclear membrane. The medication group, RGCs form, the perinuclear space increased basically normal, the chromatin distribution, margination phenomenon is not obvious, no obvious cell edema, basic membrane integrity.
Observation of 5.2 retinal NLF capillaries by electron microscopy
The normal group, the vascular wall integrity, cells arranged regularly. High intraocular pressure group, vascular wall cells damaged, basement membrane rupture, the lack of a complete vascular wall. Complete vascular wall cells in the treatment group, the endothelial cells and pericytes of mild edema and mild thickening of the basement membrane, the overall shape close to the normal group.
conclusion
1, urokinase may indirectly inhibit the apoptosis of RGCs and improve or delay the degeneration of optic nerve axons by improving the retinal microcirculation and enhancing the repair effect of optic nerve axons.
2 the importance of thrombolytic therapy in the retina is suggested for the protection of glaucomatous visual function.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R775.2

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