Boyle D L, Takemoto L J
Kansas State University, Division of Biology, Ackert Hall, Manhattan, KS 66506, USA.
Exp Eye Res. 1999 Jan;68(1):41-9. doi: 10.1006/exer.1998.0579.
The purpose of the current study was to localize the lens membrane protein, MIP 26, in nuclear fiber cells from different regions of aged normal and age-related cataractous human lenses. Adult, juvenile, fetal and embryonic nuclear regions in aged normal and age-related nuclear cataractous lenses were morphologically and biochemically characterized using the technologies of immuno-gold (5 nm) labeling, semi-thin sections (200-500 nm), serial sections, DiI staining following by photobleaching, transmission electron microscopy and spot-blot analysis. Numbers of gold particles per micron length of plasma membrane and numbers of gold particles per square micron of cytosol in the embryonic-fetal and juvenile-adult nuclear regions were quantified. Results showed that the labeling pattern of MIP 26 localized to the cytosol was unique to senescent fiber cells from age-related cataractous lenses. Numbers of gold particles per square micron of cytosol in the embryonic-fetal nucleus of age-related cataractous lenses were significantly elevated (P<0.001) above numbers from fiber cells located within the adult or juvenile nuclei of the same lens or senescent fiber cells from aged normal lenses. Some of the cytosolic labeling in cataracts was localized to lipid vesicles, while the remaining labeling was negative for the lipid specific stain DiI. Spot blot analysis demonstrated that binding of the ant-MIP 26 serum was exclusive to large molecular weight components greater than 10 kDa, and not to small molecular weight fragments of the protein. The results of the current study supply further evidence that damage to membranes occurs in senescent fiber cells during age-related nuclear cataracts, resulting in the internalization of structures containing the membrane protein MIP 26.
本研究的目的是在老年正常和年龄相关性白内障患者晶状体不同区域的核纤维细胞中定位晶状体膜蛋白MIP 26。使用免疫金（5纳米）标记、半薄切片（200 - 500纳米）、连续切片、光漂白后DiI染色、透射电子显微镜和斑点印迹分析等技术，对老年正常和年龄相关性核性白内障晶状体中的成人、青少年、胎儿和胚胎核区域进行形态学和生物化学特征分析。对胚胎 - 胎儿和青少年 - 成核区域每微米质膜长度的金颗粒数量以及每平方微米胞质溶胶中的金颗粒数量进行了定量。结果表明，MIP 26定位于胞质溶胶的标记模式是年龄相关性白内障晶状体衰老纤维细胞所特有的。年龄相关性白内障晶状体胚胎 - 胎儿核中每平方微米胞质溶胶中的金颗粒数量显著高于同一晶状体成人或青少年核内的纤维细胞或老年正常晶状体衰老纤维细胞中的金颗粒数量（P<0.001）。白内障中的一些胞质标记定位于脂质小泡，而其余标记对脂质特异性染料DiI呈阴性。斑点印迹分析表明，抗MIP 26血清的结合仅针对大于10 kDa的大分子成分，而不针对该蛋白的小分子片段。本研究结果进一步证明，在年龄相关性核性白内障形成过程中，衰老纤维细胞的膜会受到损伤，导致含有膜蛋白MIP 26的结构内化。
