本文關鍵詞：尿激酶對慢性青光眼大鼠視網(wǎng)膜神經(jīng)節(jié)細胞的保護作用　出處：《新鄉(xiāng)醫(yī)學院》2014年碩士論文　論文類型：學位論文

  更多相關文章： 凋亡 尿激酶 青光眼 局部血液循環(huán) 視網(wǎng)膜神經(jīng)節(jié)細胞

【摘要】：背景青光眼是一種常見的致盲性疾病,其發(fā)病率有相對增長趨勢。公認的唯一有效的治療方法是降低眼內壓,但眼壓正常后并不能阻止視神經(jīng)損傷的進展。視神經(jīng)損害的最終結果是視網(wǎng)膜神經(jīng)節(jié)細胞(RGCs)的凋亡,而目前視神經(jīng)節(jié)細胞保護藥物尚無確切有效的依據(jù)而應用于臨床。 目的建立大鼠青光眼壓模型,觀察尿激酶對慢性青光眼大鼠視網(wǎng)膜神經(jīng)節(jié)細胞和神經(jīng)纖維層(NFL)毛細血管內皮細胞P-JNK蛋白表達的影響；探討尿激酶對慢性青光眼視神經(jīng)的保護機制,為臨床治療提供理論依據(jù)。 方法將45只健康SD雄性大鼠,隨機分組,分別為正常組、高眼壓組和尿激酶組,每組15只,采用鞏膜上靜脈灼燒烙閉法建立慢性高眼壓青光眼模型。高眼壓組和治療組分別建立青光眼慢性高眼壓模型,正常對照組不作任何處理.用Tono-pen AVVI手持筆式眼壓計測量大鼠眼壓。在7-10天后眼壓穩(wěn)定,治療組給予腹腔注射尿激酶,正常組和高眼壓組給予等量的生理鹽水。造模1月后取材(眼球),分別處理后,行視網(wǎng)膜常規(guī)HE染色、P-JNK免疫組化和免疫熒光檢測及電鏡照相。采用SPSS13.0統(tǒng)計軟件處理數(shù)據(jù)。各組測量數(shù)據(jù)以均數(shù)士標準差(±s)表示,三組總體比較采用方差分析,其中兩組間的比較采用LSD-t檢驗,以P=0.05為標準進行比較。 結果 1眼壓測量 制模前,各組大鼠的平均眼壓在統(tǒng)計上沒有顯著性差異(P0.05)。制模后,高眼壓組、尿激酶組眼壓顯著升高,統(tǒng)計上存在顯著性差異(P0.05),1個月內眼壓無明顯的波動。在整個過程中,高眼壓組和尿激酶組分別與正常對照組相比有統(tǒng)計學的顯著性差異(P0.05)。而高眼壓組和尿激酶組相比,眼壓無統(tǒng)計學顯著性(P0.05)。 2HE染色 在正常對照組,視網(wǎng)膜外核層、內核層和神經(jīng)節(jié)細胞(RGCs)排列整齊,細胞核規(guī)則。在高眼壓組,外核層、內核層和RGCs排列疏松,不規(guī)整,其RGCs數(shù)目明顯減少。尿激酶組外核層和內核層細胞排列稍紊亂,但較高眼壓組清晰,RGCs數(shù)目較正常組略微下降,三組RGCs數(shù)總體比較,差異有統(tǒng)計學意義(P0.05)。 3P-JNK免疫組化檢測 正常組視網(wǎng)膜NLF和內顆粒層細胞基本未發(fā)現(xiàn)P-JNK蛋白陽性表達,慢性高眼壓組NLF中RGCs和血管內皮細胞及內核層細胞發(fā)現(xiàn)大量的P-JNK蛋白陽性表達,尿激酶治療組中RGCs P-JNK蛋白陽性表達明顯減少,血管內皮細胞和內顆粒層未發(fā)現(xiàn)P-JNK蛋白陽性表達。三組視網(wǎng)膜平均灰度值總體比較,差異有統(tǒng)計學意義(P0.05)。高眼壓組P-JNK平均灰度值與正常組比較,差異有統(tǒng)計學意義(P0.05)；尿激酶治療組與其高眼壓組組比較,差異有統(tǒng)計學意(P0.05),尿激酶治療組與正常組比較,差異有統(tǒng)計學意(P0.05)。 4P-JNK免疫熒光檢測 正常組視神經(jīng)鞘膜外纖維細胞基本上未發(fā)現(xiàn)P-JNK蛋白陽性表達,慢性高眼壓組有較少的P-JNK蛋白陽性表達,尿激酶治療者P-JNK蛋白陽性表達較高眼壓組略有增強。三組視網(wǎng)膜神經(jīng)節(jié)細胞層P-JNK平均熒光強度總體比較,差異有統(tǒng)計學意義(P0.05)。高眼壓組P-JNK平均灰度值與正常組比較,差異有統(tǒng)計學意義(P0.05)；尿激酶治療組與其高眼壓組組比較,差異有統(tǒng)計學意(P0.05),尿激酶治療組與正常組比較,差異有統(tǒng)計學意(P0.05)。 5電鏡結果 5.1RGCs的電鏡觀察 正常組,RGCs核形態(tài)規(guī)則,核周間隙正常,其染色質分布均勻,沒有發(fā)現(xiàn)邊集現(xiàn)象。細胞器沒有看見太明顯的水腫現(xiàn)象。質膜完整。高眼壓組,RGCs形態(tài)不規(guī)則,核周間隙明顯增寬,其染色質分布不均勻,有邊集現(xiàn)象。細胞器呈現(xiàn)高度的水腫現(xiàn)象伴不完整的質膜。用藥組,RGCs核形態(tài)規(guī)則,核周間隙增基本正常,其染色質分布均勻,邊集現(xiàn)象不明顯,細胞器未見很明顯的水腫現(xiàn)象,質膜基本完整。 5.2視網(wǎng)膜NLF毛細血管的電鏡觀察 正常組,血管壁完整,細胞排列規(guī)整。高眼壓組,血管壁細胞遭到嚴重破壞,基底膜破裂,缺乏一個完整的血管壁。治療組的完全血管壁細胞、內皮細胞和周細胞輕度水腫,基底膜輕度增厚,整體形狀接近正常組。 結論 1尿激酶可能是通過改善視網(wǎng)膜的微循環(huán)和增強視神經(jīng)軸突的修復作用,間接抑制RGCs的凋亡及改善或延遲視神經(jīng)軸突變性。 2提示視網(wǎng)膜局部的溶栓治療對青光眼視功能保護的重要性。
[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.

【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R775.2

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