人晶状体细胞膜流动性的年龄相关性改变及可溶性晶状体蛋白的调节作用

发布时间：2018-03-25 12:38

本文选题：衰老　切入点：人晶状体　出处：《复旦大学》2010年博士论文

【摘要】： 人的晶状体是一个特殊的器官,其赤道部的晶状体上皮细胞(lens epithelial cells, LECs)不断分化形成新的晶状体纤维,并向核区移行,在此过程中纤维细胞的细胞核和细胞器不断退化消失,仅留下透明的细胞质,从而形成了几乎不存在蛋白质更新的核区。这一核区担负着人眼调节的主要形变任务,同时它又是翻译后修饰的主要区域,双重的负荷使其容易受到各种年龄效应的影响。随着年龄的增长,晶状体组织的硬度明显增加,尤其是核区,从而加速了老视和年龄相关性白内障的发生。由于晶状体核区的含水量并未发生明显的改变,这种硬度的增加与核区内纤维的累积关系不大。研究表明,到了中年,人晶状体中50%原本可溶性的蛋白质变得不溶,从而使晶状体的硬度增加；而对红细胞和胆管细胞的研究表明,随着年龄的增长其细胞膜的流动性会不断下降。那么人晶状体细胞膜会发生什么样的年龄相关性改变呢,变的更致密还是流动性增加?它的改变是不是也是晶状体硬度增加的原因之一呢?本研究通过观察分析人晶状体细胞膜流动性的年龄相关性改变,探索促使其变化的可能原因,来明确这些疑问。目的观察人晶状体细胞膜流动性的年龄相关性改变。方法23对人晶状体,22-83岁。每对中一个晶状体制作冠状位切片,取中央部位的切片进行6-酰基-2-二甲酰氨基萘(6-acyl-2-dimethylaminonapthalene, Laurdan)染色,并用双光子共聚焦显微镜沿切片最长轴连续采集图像,用软件计算转换得到GP (Generalized Polarization)图,把来自同一切片的GP图像用ImageJ-MosaicJ软件组合后,分别测量中心点两侧距离为0.75,2,3,3.5,4,4.5mm处的GP值。每对中另一个晶状体用模具解剖成外部、屏障部、内部和核心部,进行脂质提取,并用质谱分析每个部分的胆固醇含量。结果20-40岁时,晶状体从外到内的细胞膜流动性几乎恒定；随着年龄的增加,人晶状体中央的细胞膜流动性逐渐增加,而距中心3.5mm以外的细胞膜流动性改变不明显。胆固醇的含量没有明显的年龄性或区域性改变。结论人晶状体核区的细胞膜流动性随年龄增加而增加,这与胆固醇含量的变化并不相关。目的分析人晶状体细胞膜流动性与可溶性晶状体蛋白含量改变的相关性。方法3对晶状体,年龄分别为31,40,63岁,每对中的一个晶状体直接制作冠状位切片,另一个晶状体则在50℃加热20小时后制作冠状位切片,切片的染色观察如第一部分。所有切片后剩余的组织用模具解剖获得核心部位组织,用凝胶过滤HPLC分析可溶性晶状体蛋白含量。另取一对38岁的晶状体,一个晶状体用模具获得直径6mm以内的核组织,均分成5块,另一个晶状体进行连续切片,以l块组织和3个切片为一组,分别在50℃加热0,4,8,16和24 h,分析可溶性晶状体蛋白含量改变,并测量各组切片的GP值改变。结果50℃加热后,晶状体内部细胞膜流动性显著增加,同时α,α的高分子聚集物(high molecular weight, HMW)和β晶状体蛋白的含量下降。随着加热时间的延长,晶状体内部的GP值呈现与可溶性晶状体蛋白含量一致的下降。结论晶状体细胞膜流动性的改变与可溶性晶状体蛋白含量的变化可能相关。目的研究晶状体细胞膜流动性与可溶性晶状体蛋白是否存在直接联系。方法用人晶状体内含量最高的磷脂成分DHSM制作0.1mg/ml囊泡溶液,并在实验开始前1小时加入Laurdan标记。分别配制12.5,5,2.5,1.25mg/ml的α,p,γ晶状体蛋白溶液,各取10μL与240μL囊泡混合后,最终晶状体蛋白浓度分别为0.5,0.2,0.1,0.05mg/ml,结合半小时后,用荧光计测量各组GP值变化。结果随着α和β晶状体蛋白浓度的增加,GP值逐渐升高,膜的流动性下降,而γ晶状体蛋白浓度改变对GP值没有影响。结论α和β晶状体蛋白能够影响晶状体细胞膜的流动性,而γ晶状体蛋白不具备该功能。
[Abstract]:The lens is a special organ, the equator of the lens epithelial cells (lens epithelial cells, LECs) to form a new lens fiber differentiation, and migration to the nucleus, nucleus and organelles of fibroblasts during degradation disappeared, leaving only the transparent cytoplasm, thus forming almost there is no protein turnover in the nuclear area. This area bears the main task of nuclear deformation eye adjustment, at the same time it is the main area of post-translational modifications, the double load makes it vulnerable to influence of various age effect.
With the increase of age, the lens tissue hardness increased significantly, especially in the nuclear area, thus accelerating the presbyopia and age related cataract. The lens nucleus water content did not change significantly, the Cumulative little relation to increase the hardness and the nucleus. The results show that, to the middle-aged people 50% the soluble protein in the lens become insoluble, so that the lens increases the hardness of; of red blood cells and bile duct cells showed that with the increase of the age of the liquidity of the cell membrane will continue to decline.
Then the human lens cell membrane occurs age-related what kind of change, become more dense or more fluid? It is one of the reasons is the change of lens hardness increase? Age related changes in the study through observation and analysis of cell membrane fluidity of human lenses, to explore the possible reasons for the change, to answer these questions.
Objective To observe the age-related changes in the membrane fluidity of human lens cells.
Methods 23 lens, 22-83 years old. Each pair in a lens made of coronal sections from the central part of the section for 6- -2- two benzoyl amino acyl naphthalene (6-acyl-2-dimethylaminonapthalene, Laurdan) staining, and two-photon confocal microscopy sections along the longest axis continuous image acquisition, obtained by software (GP Generalized Polarization) diagram, the GP image from the same slice with ImageJ-MosaicJ software combination, were measured on both sides of center distance of 0.75,2,3,3.5,4,4.5mm at GP. Another lens in each pair with external mold anatomy, Department of internal department and barrier, the core, of lipid extraction, and using mass spectrometry analysis of cholesterol content in each part.
Results the age of 20-40, the cell membrane of lens from the outside to the inside of a nearly constant flow; with the increase of age, the cell membrane fluidity of human lens central gradually increased, and from outside the cell membrane 3.5mm mobility change is not obvious. No cholesterol content significant age or regional change.
Conclusion the cell membrane fluidity in the nucleus of human lens increases with age, which is not related to the change of cholesterol content.
Objective to analyze the correlation between the fluidity of human lens cell membrane and the change of soluble lens protein content.
Methods 3 lens, aged 31,40,63 years old, a lens in each pair are directly made of coronal slices, another lens is heated at 50 DEG C for 20 hours after the production of coronal slices, slices were observed as the first part. All remaining after the biopsy tissue obtained by die core parts of anatomical organization, analysis the content of soluble lens proteins by gel filtration HPLC. Another 38 year old lens diameter 6mm within the nuclear organization to obtain a lens mold, were divided into 5 blocks, another lens serial sections in L block and 3 sections as a group, were heated at 50 DEG C and 24 0,4,8,16 h, changes of soluble protein contents were determined, and the measured sections of the change of GP value.
Results 50 DEG C after heating, increase liquidity, the interior of the lens cell membrane and alpha, alpha polymer aggregates (high molecular weight, HMW) content decreased and crystallin. With the prolongation of heating time, the interior of the lens GP value decreased and the content of soluble protein of the lens.
Conclusion the changes in the fluidity of the lens cell membrane may be related to the change of the soluble lens protein content.
Objective to study the direct relationship between the fluidity of the lens cell membrane and the existence of soluble lens protein.
Methods DHSM with phospholipid crystals the highest content of 0.1mg/ml vesicles, and before the experiment began 1 hours. 12.5,5,2.5,1.25mg/ml were prepared with Laurdan labeled alpha crystallin, P, solution, the 10 L and 240 L vesicles after mixing, the final lens protein concentration was 0.5,0.2,0.1,0.05mg/ml. With half an hour after the value changes were measured by fluorescence GP.
Results with the increase of the concentration of alpha and beta lens protein, the GP value increased and the membrane fluidity decreased, while the concentration change of gamma lens protein had no effect on the GP value.
Conclusion alpha and beta crystallin can affect the fluidity of the lens cell membrane, while the gamma lens protein does not have the function.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R776.1

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