【摘要】：白內障是一種發(fā)病率、致盲率均居當今國內外眼病之首的疾病，嚴重影響患者的視力健康和生活質量。研究其發(fā)病機制和藥物防治，具有重要的意義和價值。流行病學及實驗室研究表明，氧化損傷是其發(fā)生發(fā)展的主要機制之一。氫氣是一種優(yōu)質的抗氧化劑，可選擇性中和強毒性活性氧自由基，具有干擾自由基的生成，清除已生成的自由基，調節(jié)細胞代謝活性，無毒副作用等優(yōu)點。硒性白內障發(fā)病機制主要為氧化損傷，本實驗通過建立硒性白內障動物模型來初步研究氫飽和生理鹽水對亞硒酸鈉性白內障的作用機制，為臨床藥物治療白內障提供新思路。 目的： 初步研究氫飽和生理鹽水對SD大鼠亞硒酸鈉性白內障的影響及相關機制。 方法： 1氫飽和生理鹽水對大鼠硒性白內障作用的初步觀察 選用7日齡健康SD大鼠68只，根據(jù)體重按隨機數(shù)字表法把動物隨機分為6組。分別為正常對照組（A，n=12），白內障組（B，n=12），氫水預防組（C，n=12），氫水全程治療組（D，n=12），氫水晚期治療組（E，n=12），氫水組（F，n=8）。白內障組、氫水預防組、氫水全程治療組和氫水晚期治療組大鼠12日齡按25μmol/kg體重單次頸背部皮下注射2mmol/L亞硒酸鈉生理鹽水。氫水預防組大鼠8日齡～12日齡腹腔注射氫飽和生理鹽水5ml/kg，每日1次，連續(xù)5d；氫水全程治療組大鼠8日齡～17日齡腹腔注射氫飽和生理鹽水5ml/kg，每日1次，連續(xù)10d；氫水晚期治療組大鼠17日齡～21日齡腹腔注射氫飽和生理鹽水5ml/kg，每日1次，連續(xù)5d；氫水組大鼠8日齡～17日齡腹腔注射氫飽和生理鹽水5ml/kg，每日1次，連續(xù)10d。于26日齡裂隙燈顯微鏡下觀察大鼠晶狀體混濁程度，測定晶狀體中水溶性蛋白含量，SOD活性及GSH、MDA含量。所得數(shù)據(jù)進行統(tǒng)計學分析，以確定可能抑制白內障的作用時間。 2氫飽和生理鹽水對大鼠硒性白內障的影響機制的研究 根據(jù)第一部分實驗結果，進一步研究氫飽和生理鹽水對大鼠硒性白內障的影響機制。選用7日齡健康SD大鼠60只，根據(jù)體重按隨機數(shù)字表法把動物隨機分為3組。分別為正常對照組（A，n=20），白內障組（B，n=20），氫水全程治療組（C，n=20）。白內障組和氫水全程治療組大鼠12日齡按25μmol/kg體重單次頸背部皮下注射2mmol/L亞硒酸鈉生理鹽水。氫水全程治療組大鼠8日齡～17日齡腹腔注射氫飽和生理鹽水5ml/kg，每日1次，連續(xù)10d。于26日齡裂隙燈顯微鏡下觀察大鼠晶狀體混濁程度，測定晶狀體中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及MDA、GSH、總巰基含量，以及視網(wǎng)膜中SOD、CAT、Gpx、GR活性及GSH、MDA含量。所得數(shù)據(jù)進行統(tǒng)計學分析。 結果： 1氫飽和生理鹽水對大鼠硒性白內障作用的初步觀察 (1)晶狀體混濁程度的觀察： 正常對照組和氫水組大鼠在整個實驗過程中晶狀體始終透明均為0級；白內障組16.67%（4/24）晶狀體Ⅴ級混濁，83.33%（20/24）晶狀體Ⅵ級混濁；氫水預防組25%（6/24）晶狀體Ⅴ級混濁，75%（18/24）晶狀體Ⅵ級混濁；氫水全程治療組33.33%（8/24）晶狀體Ⅲ級混濁，41.67%（10/24）晶狀體Ⅳ級混濁，16.67%（4/24）晶狀體Ⅴ級混濁，8.33%（2/24）晶狀體Ⅵ級混濁；氫水晚期治療組29.17%（7/24）晶狀體Ⅴ級混濁，70.83%（17/24）晶狀體Ⅵ級混濁。氫水預防組、氫水晚期治療組與白內障組比較，晶狀體混濁程度無明顯差異，統(tǒng)計學檢驗結果顯示差異無統(tǒng)計學意義(χ~2=1.06，P0.05)。氫水全程治療組與白內障組比較，晶狀體混濁的發(fā)生發(fā)展相對延緩，混濁程度明顯低于白內障組，統(tǒng)計學檢驗結果顯示差異有統(tǒng)計學意義(χ~2=30.48，P0.05)。 (2)晶狀體各項生化指標比較： 各組晶狀體中水溶性蛋白含量，SOD活性及GSH、MDA含量比較，白內障組、氫水預防組、氫水晚期治療組較正常對照組、氫水組晶狀體中水溶性蛋白含量，SOD活性及GSH含量均明顯下降(P0.05)，氫水全程治療組水溶性蛋白含量，SOD活性及GSH含量下降不明顯，且明顯高于白內障組(P0.05)，差異有統(tǒng)計學意義，但氫水預防組、氫水晚期治療組水溶性蛋白含量，SOD活性及GSH含量較白內障組無明顯改變(P0.05)；白內障組、氫水預防組、氫水晚期治療組較正常對照組、氫水組晶狀體中MDA含量明顯升高(P0.05)，氫水全程治療組MDA含量升高不明顯，且明顯低于白內障組(P0.05)，差異有統(tǒng)計學意義，但氫水預防組、氫水晚期治療組MDA含量較白內障組無明顯改變(P0.05)。氫水組與正常對照組比較，生化指標無改變(P0.05)。 2氫飽和生理鹽水對大鼠硒性白內障的影響機制的研究 (1)晶狀體混濁程度的觀察： 正常對照組大鼠在整個實驗過程中晶狀體始終透明均為0級；白內障組20%（8/40）晶狀體Ⅳ級混濁，25%（10/40）晶狀體Ⅴ級混濁，55%（22/40）晶狀體Ⅵ級混濁；氫水全程治療組5%（2/40）晶狀體Ⅱ級混濁，25%（10/40）晶狀體Ⅲ級混濁，45%（18/40）晶狀體Ⅳ級混濁，15%（6/40）晶狀體Ⅴ級混濁，10%（4/40）晶狀體Ⅵ級混濁。氫水全程治療組與白內障組比較，晶狀體混濁的發(fā)生發(fā)展相對延緩，混濁程度明顯低于白內障組，統(tǒng)計學檢驗結果顯示差異有統(tǒng)計學意義(χ~2=44.06，P0.05)。 (2)晶狀體各項生化指標比較： 各組晶狀體中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及MDA、GSH、總巰基含量比較，白內障組較正常對照組晶狀體中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、總巰基含量均明顯下降(P0.05)，氫水全程治療組水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、總巰基含量下降不明顯，且明顯高于白內障組(P0.05)，，差異有統(tǒng)計學意義；白內障組較正常對照組晶狀體中MDA含量明顯升高(P0.05)，氫水全程治療組MDA含量升高不明顯，且明顯低于白內障組(P0.05)，差異有統(tǒng)計學意義。 (3)視網(wǎng)膜各項生化指標比較： 各組大鼠視網(wǎng)膜中SOD、CAT、Gpx、GR活性及GSH、MDA含量比較，正常對照組、白內障組、氫水全程治療組各項生化指標無明顯區(qū)別，差異無統(tǒng)計學意義(P0.05)。 結論： 1.氫飽和生理鹽水可減輕亞硒酸鈉對晶狀體的氧化損傷，對亞硒酸鈉性白內障有一定的抑制作用，但無明顯的預防作用，對于已形成的白內障無逆轉作用，對晶狀體無明顯的毒性作用。 2.氫飽和生理鹽水可以提高晶狀體組織中水溶性蛋白含量，SOD、CAT、Gpx、GR、GST活性及GSH、總巰基含量，減少MDA生成，從而減輕晶狀體的氧化損傷，對亞硒酸鈉性白內障的發(fā)生和發(fā)展有一定的抑制和延緩作用。 3.氫飽和生理鹽水對大鼠視網(wǎng)膜正常氧化還原狀態(tài)的平衡無明顯影響。 創(chuàng)新點：首次探討了氫飽和生理鹽水對大鼠硒性白內障抑制作用及相關機制,為臨床藥物治療白內障提供新思路。
[Abstract]:The incidence of cataract is a disease rate, and the rate of blindness is the first disease at home and abroad, which seriously affects the health and quality of life of the patients. It is of great significance and value to study the pathogenesis and drug control of the patients. The epidemiological and laboratory studies show that the oxidative damage is one of the main mechanisms of its development. High quality antioxidants, which can selectively neutralize strong toxic reactive oxygen radicals, interfere with the generation of free radicals, remove the generated free radicals, regulate cell metabolic activity, and have no side effects. The pathogenesis of selenite cataract is mainly oxidative damage. In this experiment, a selenium induced cataract animal model was developed to study the initial hydrogen satiety. And the physiological mechanism of sodium selenite in the treatment of selenite induced cataract, so as to provide new ideas for clinical medicine in the treatment of cataract.
Objective:
Objective to investigate the effect of hydrogen saturated saline on selenite induced cataract in SD rats and its mechanism.
Method:
Effect of 1 hydrogen saturated saline on selenium induced cataract in rats
68 healthy SD rats of 7 days of age were randomly divided into 6 groups according to their weight and random numbers. They were normal control group (A, n=12), cataract group (B, n=12), C (n=12), whole hydrogen water treatment group (D, n=12), hydrogen water advanced treatment group (E, n=12), hydrogen water group, cataract group, hydrogen water prevention group and whole hydrogen water treatment group. Rats in the late treatment group of hydrogen and water were injected with 2mmol/L sodium selenite subcutaneous injection at the 12 day of 25 mol/kg body weight on the back of the neck. The hydrogen water prevention group was intraperitoneally injected with hydrogen saturated saline 5ml/kg at the age of 8 to 12 days of age, 1 times a day, and continuous 5D, and hydrogen saturated saline 5ml/ was injected into the group of rats at the age of 8 days to 17 days of hydrogen water treatment group, 5ml/ Kg, 1 times a day and continuous 10d; the rats in the late treatment group of hydrogen and water were intraperitoneally injected with hydrogen saturated saline 5ml/kg at the age of 17 to 21 days of age, 1 times a day and continuous 5D. The hydrogen saturated rats were intraperitoneally injected with hydrogen saturated saline 5ml/kg, 1 times a day, and observed the degree of lens opacities in the rat lens under the 26 day Gap light microscope, and the degree of lens opacity was observed under the 26 day Gap light microscope. The contents of water-soluble protein, SOD activity, GSH and MDA in the crystalline lens were determined by statistical analysis.
Effect of 2 hydrogen saturated saline on selenium induced cataract in rats
According to the results of the first part of the experiment, we further studied the mechanism of the effect of hydrogen saturated saline on selenium induced cataract in rats. 60 healthy SD rats of 7 day old were randomly divided into 3 groups according to the weight according to the random number table. They were normal control group (A, n=20), cataract group (B, n=20), C (n=20), cataract group and cataract group. The rats were injected with 2mmol/L sodium selenite under the neck and back of 25 mol/kg body weight at 12 days of age. The rats were injected with hydrogen saturated saline 5ml/kg from 8 days to 17 days of age of hydrogen water treatment group, 1 times a day, and the degree of crystalline lens opacities was observed under the 26 day old slit lamp microscope, and the crystal shape was measured. The content of water-soluble protein in body, SOD, CAT, Gpx, GR, GST activity and MDA, GSH, total sulfhydryl content, and SOD in the retina, CAT, Gpx, GR activity and GSH, with statistical analysis.
Result:
Effect of 1 hydrogen saturated saline on selenium induced cataract in rats
(1) the observation of the degree of lens opacity:
In the normal control group and the hydrogen water group, the MICROTEK body was all the 0 level in the whole experiment, the cataract group 16.67% (4/24) lens V turbidity, 83.33% (20/24) lens VI turbidity, the hydrogen water prevention group 25% (6/24) lens V grade turbidity, 75% (18/24) crystal VI turbidity, and the 33.33% (8/24) lens III of the hydrogen water treatment group. Turbidity grade, 41.67% (10/24) lens IV turbidity, 16.67% (4/24) lens V turbidity, 8.33% (2/24) lens VI turbidity, 29.17% (7/24) lens V turbidity in late hydrogen water treatment group and 70.83% (17/24) lens VI turbidity. Hydrogen water prevention group, hydrogen water advanced treatment group and cataract group, the degree of lens opacities had no significant difference. The statistical test showed that the difference was not statistically significant (x ~2=1.06, P0.05). Compared with the cataract group, the development and development of lens opacities were relatively delayed, and the degree of turbidity was significantly lower than that of the cataract group. The statistical results showed that the difference was statistically significant (x ~2=30.48, P0.05).
(2) the comparison of the biochemical indexes of the lens:
The content of water-soluble protein, SOD activity and GSH, MDA content, cataract group, hydrogen water prevention group, and hydrogen water advanced treatment group were compared with normal control group. The water soluble protein content, SOD activity and GSH content in the hydrogen water group were significantly decreased (P0.05), the content of water soluble protein in the whole treatment group of hydrogen water, the decrease of SOD activity and GSH content in the hydrogen water treatment group. It was not obvious, and obviously higher than the cataract group (P0.05), the difference was statistically significant, but the water soluble protein content, the SOD activity and the GSH content in the hydrogen water treatment group were not significantly changed (P0.05), the cataract group, the hydrogen water prevention group, the late hydrogen water treatment group were compared with the normal control group, and the MDA content in the hydrogen water group was obvious. Increase (P0.05), MDA content in the hydrogen water treatment group increased not significantly, and obviously lower than the cataract group (P0.05), the difference was statistically significant, but the hydrogen water prevention group, the late hydrogen water treatment group MDA content than the cataract group was not significantly changed (P0.05). Hydrogen water group and normal control group, biochemical indexes did not change (P0.05).
Effect of 2 hydrogen saturated saline on selenium induced cataract in rats
(1) the observation of the degree of lens opacity:
In the normal control group, the MICROTEK body was all the 0 level in the whole experiment, the cataract group 20% (8/40) lens IV turbidity, 25% (10/40) lens V grade turbidity, 55% (22/40) lens VI turbidity, 5% (2/40) lens II turbidity, 25% (10/40) lens III turbidity, 45% (18/40) lens IV mixing in the whole course of hydrogen water treatment group. Turbidity, 15% (6/40) lens V grade turbidity, 10% (4/40) lens VI turbidity. Compared with cataract group, the occurrence and development of lens opacities were relatively delayed, and the degree of turbidity was significantly lower than that of cataract group. Statistical results showed that the difference was statistically significant (x ~2=44.06, P0.05).
(2) the comparison of the biochemical indexes of the lens:
The content of water-soluble protein, SOD, CAT, Gpx, GR, GST activity and MDA, GSH, total sulfhydryl content, the content of water-soluble protein in cataract group compared with that of normal control group, SOD, CAT, Gpx, GR, GST activity and total sulfhydryl content decreased significantly. And GSH, the total sulfhydryl content decreased not obviously, and obviously higher than the cataract group (P0.05), the difference was statistically significant. The content of MDA in the cataract group was significantly higher than that in the normal control group (P0.05). The increase of MDA content in the whole hydrogen water treatment group was not obvious, and it was significantly lower than the cataract group (P0.05), the difference was statistically significant.
(3) the comparison of the biochemical indexes of the retina:
The activities of SOD, CAT, Gpx, GR and GSH and MDA in the retina of rats were compared. There was no significant difference in the biochemical indexes between the normal control group, the cataract group and the whole treatment group of hydrogen water (P0.05).
Conclusion:
1. hydrogen saturated saline can reduce the oxidative damage of sodium selenite to the lens and inhibit the sodium selenite cataract, but it has no obvious preventive effect. It has no reverse effect on the formed cataract and has no obvious toxic effect on the lens.
2. hydrogen saturated saline can improve the content of water-soluble protein in lens tissue, SOD, CAT, Gpx, GR, GST activity and GSH, total sulfhydryl content, reduce MDA generation, thus reduce the oxidative damage of the lens, and have certain inhibition and delay effect on the occurrence and development of sodium selenite cataract.
3. hydrogen saturated saline had no significant effect on the balance of retinal redox state in rats.
Innovation: The inhibitory effect of hydrogen-saturated saline on selenium-induced cataract in rats and its related mechanisms were discussed for the first time, which provided a new idea for clinical treatment of cataract.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R776.1

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