褪黑素对抑制过氧化氢诱导大鼠白内障形成的实验研究

发布时间：2018-05-13 23:28

本文选题：褪黑素 + 白内障　；参考：《山东大学》2010年硕士论文

【摘要】： 研究目的：白内障是一致盲眼病,氧化损伤是诱发白内障的重要因素。褪黑素(MLT)是松果体分泌的一种吲哚类激素,是人体内强有效的自由基清除剂及抗氧化剂。本课题应用褪黑素作用于离体氧化损伤的大鼠晶状体,通过体外抗氧化模型实验,检测其抗氧化能力,探讨褪黑素对过氧化氢诱导大鼠白内障形成的抑制作用,为寻求较为理想的抗氧化药物提供理论依据。研究方法：体外取出发育正常的Wistar大鼠晶状体,按随机原则分为以下三组：A组为空白对照组,B组为过氧化氢(H2O2)处理组,C组为在B组的基础上加入不同浓度的MLT,使MLT终浓度分别为10(C1组)、30(C2组)、50(C3组)、100(C4组)、200(C5组)μmol／L。体外培养24h后,观察各组晶状体的混浊程度,采用Image-ProPlus(IPP)6.0软件分析晶状体下黑色条带的平均光密度值；酶标仪测定各组晶状体组织的蛋白含量、还原型谷胱甘肽(GSH)、丙二醛(MDA)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)含量。行HE染色倒置显微镜下观察各组晶状体上皮细胞的形态。研究结果： 1.大鼠晶状体混浊程度的观察及定量分析培养结束后,裂隙灯下可见,A组晶状体保持透明,晶状体后灰线清晰可见；B组晶状体全混呈白色,晶状体后灰线模糊不清；C组晶状体的透明性均高于B组,晶状体后灰线呈不同程度的模糊。平均光密度值分析,C组晶状体平均光密度值高于B组(P0.05)。C组结果显示,C3组与C4组平均光密度值较高,晶状体透明性较高,组间差异无统计学意义(P0.05),其它各组间平均光密度值差异均有统计学意义(P0.05)。 2.大鼠晶状体生化指标的测定培养结束后,C组大鼠晶状体组织中的GSH、T-AOC、SOD、CAT含量高于B组(P0.05),在C组中,C3组大鼠晶状体组织中的GSH、T-AOC、CAT活性最高(P0.05), C3组与C4组SOD含量差异无统计学意义(P0.05)。C组大鼠晶状体组织中MDA含量低于B组(P0.05),而C3、C4、C5组组间MDA含量差异无统计学意义(P0.05)。 3.光镜下各组晶状体组织的细胞形态学观察HE染色后,光镜下可见,空白对照组晶状体前囊膜连续光滑,晶状体上皮细胞线状排列,核呈长杆状,晶状体皮质纤维层排列规整。H202处理组晶状体前囊膜不连续,晶状体上皮细胞有增殖,排列紊乱,部分晶状体上皮细胞向深层组织移行,细胞体积变大,胞核多形。MLT10μmol／L组晶状体前囊膜完整,晶状体上皮细胞增殖,形态呈长杆状,排列呈多层,多位于较深层次；MLT30μmol/L组部分晶状体上皮细胞增殖,失去原来整齐的单层排列,出现拥挤现象,晶状体皮质纤维层排列规整；MLT50μmol／L组晶状体上皮细胞线状排列,间隙较一致,胞核呈长杆状,形态较一致。随着MLT浓度的增加,可见晶状体上皮细胞线状排列,间隙较一致,少量细胞位于较深层,晶状体皮质纤维层排列紧密而规整,但是MLT100μmol／L组有囊泡形成。研究结论：在H202存在的条件下,未加入MLT,大鼠晶状体全混呈白色。在H2O2存在的条件下,加入MLT,大鼠晶状体的透明度明显高于H2O2处理组。MLT及其被氧化后的多级代谢产物具有较强的抗氧化作用,抵抗H2O2对晶状体的氧化损伤。本实验中加入MLT体外培养的大鼠晶状体GSH、T-AOC、SOD及CAT含量高于H2O2处理组,而MDA含量低于H2O2处理组,说明MLT组抗氧化能力提高。在加入MLT各组间,从MLT 10μmol/L至MLT50μmol/L,随着浓度的增加,其抗氧化能力越强,MLT在浓度为50μmol/L时即能发挥有效作用。光镜下观察大鼠晶状体上皮细胞的形态,H2O2处理组晶状体前囊膜不完整,晶状体上皮细胞排列紊乱,呈多层,细胞间隙增大,胞核多形；而MLT组上皮细胞破坏程度比H2O2处理组轻,MLT50μmol/L组晶状体上皮细胞呈单层线状排列,细胞间隙较一致,胞核呈长杆状,形态更接近空白对照组,这些实验结果说明MLT可提高氧化损伤晶状体的抗氧化能力,发挥了很强的保护晶状体、维持晶状体透明性的作用。
[Abstract]:The purpose of the study is:
Cataract is an unanimous blind eye disease, and oxidative damage is an important factor in inducing cataract. Melatonin (MLT) is an indole hormone secreted by the pineal body. It is a strong effective free radical scavenger and antioxidant in the human body. This subject applies melatonin to the lens of rats with oxidative damage in vitro and tests the antioxidant model in vitro. To explore the inhibitory effect of melatonin on the formation of cataract induced by hydrogen peroxide in rats, and to provide a theoretical basis for seeking more ideal antioxidation drugs.
Research methods:
The lens of Wistar rats developed in vitro was divided into three groups according to the random principle: group A was blank control group, group B was hydrogen peroxide (H2O2), and C group was added with different concentrations of MLT on the basis of group B, and the final concentration of MLT was 10 (C1 group), 30 (C2 group), 50 (C3 group), 100 (C4 group), 200 (Group)). The degree of turbidity in the lenses of each group was observed. The average optical density of the black strip under the lens was analyzed by Image-ProPlus (IPP) 6. The protein content of the lens tissue in each group was measured by the enzyme labeling instrument, and the glutathione (GSH), the malondialdehyde (MDA), the total antioxidant energy (T-AOC), the superoxide dismutase (SOD) and the catalase (CAT) content were also measured. The morphology of lens epithelial cells was observed under inverted microscope with HE staining.
The results of the study:
After the observation and quantitative analysis of the degree of lens opacities in 1. rats, the lens under the slit lamp was visible, the lens of the A group remained transparent and the posterior ash line of the lens was clearly visible; the B group was completely mixed with white, and the posterior ash line of the lens was blurred; the transparency of the C group was higher than that of the B group, and the posterior ash line of the lens was blurred in varying degrees. The mean light density value of C group was higher than that of group B (P0.05).C group. The mean light density of C3 group and C4 group was higher, and the transparency of lens was higher. There was no statistical difference between groups (P0.05), and the difference of average light density between the other groups had statistical significance (P0.05).
The content of GSH, T-AOC, SOD, CAT in the lens tissue of group C rats was higher than that of group B (P0.05) after the determination of the biochemical indexes of the lens. In group C, GSH, T-AOC, and CAT activity in the lens tissues of group C3 rats were the highest. In group B (P0.05), there was no significant difference in MDA content between groups C3, C4 and C5 (P0.05).
The morphology of the lens tissue in each group was observed under the 3. light microscope after HE staining. In the blank control group, the anterior capsule of the lens was smooth, the lens epithelial cells were arranged linearly, the nucleus was long rod like, the fibrous layer arranged in the lens cortex was discontinuous in the pre lens capsule of the.H202 treatment group, and the lens epithelial cells proliferated and arranged turbulence. In disorder, the epithelial cells of part of the lens moved to the deep tissue, the cell volume became larger, the nucleus of the nucleus polymorphs.MLT10 Mu mol / L was complete, the epithelial cells of the lens proliferated, the morphology was long rod like, and the arrangement was multi-layer and more in the deeper level, and the MLT30 mu mol/L group partial crystalline epithelial cells proliferated and lost the original neat monolayer arrangement. The fibrous layer of the lens cortex was arranged regularly, and the lens epithelial cells in MLT50 Mu mol / L were arranged linearly, with a relatively uniform space, and the nuclei were long rod-shaped, and the morphology was more consistent. With the increase of MLT concentration, the lens epithelial cells were arranged linearly, the gaps were more consistent, a small number of cells were located in the deeper layer, and the lens cortical fibrous layer arranged. Tight and regular, but vesicles formed in the MLT100 mol / L group.
The conclusions are as follows:
Under the presence of H202, the rat lens was mixed with white without MLT. Under the condition of H2O2, the transparency of the lens was obviously higher than that of the H2O2 treatment group.MLT and the multilevel metabolites after the oxidation, which resisted the oxidative damage of the H2O2 to the lens. In this experiment, MLT in vitro was added to the experiment. The content of GSH, T-AOC, SOD and CAT in cultured rat lens is higher than that in H2O2 treatment group, and the content of MDA is lower than that of H2O2 treatment group. It shows that the antioxidant capacity of MLT group is improved. The antioxidant capacity of MLT group is stronger with the increase of MLT 10 mu mol/L to MLT50. The morphology of the lens epithelial cells in the rats was observed. The anterior capsule of the H2O2 treatment group was incomplete, the lens epithelial cells were arranged in disorder, the lens epithelial cells were multilayered, the cell space was enlarged, and the nucleus was polymorphic. The damage degree of the epithelial cells in the group MLT was lighter than that of the H2O2 treatment group. The epithelial cells in the group of MLT50 mu mol/L were arranged in a single linear arrangement and the cell space was more consistent. The nucleation was long rod like, and the morphology was closer to the blank control group. These results showed that MLT could improve the antioxidant capacity of the oxidative damaged lens, and played a strong role in protecting the lens and maintaining the transparency of the lens.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1

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