钙介导的解体球化过程中单个晶状体纤维透光率的变化。

Bhatnagar A, Dhir P, Wang L F, Ansari N H, Lo W, Srivastava S K

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647, USA.

Invest Ophthalmol Vis Sci. 1997 Mar;38(3):586-92.

PURPOSE

The purpose of this study was to examine changes in the light transmission through single cortical fibers isolated from the rat lens during the process of disintegrative globulization.

METHODS

Single cortical fibers were isolated from adult rat lens by treatment with trypsin in a solution containing 10 mM HEPES, 10 mM EDTA, and 280 mM sucrose (pH 7.4, 300 to 310 mOsm) HEPES-EDTA-sucrose (HES) solution. The isolated fibers were illuminated by a white light source, and the light transmission through the fibers was collected by a charge-coupled device camera and quantified by digital image analysis. In some experiments, thin sections of fixed lens cells were examined using transmission electron microscopy.

RESULTS

Enzymatic dissociation of the lens yielded elongated fibers, which, in the presence of Ringer's solution (containing 2 mM Ca2+), underwent disintegrative globulization. Measurements of light transmission through elongated fibers suspended in HES solution showed maximal transmission at the center of the fiber. Exposure of the cortical fibers to Ringer's solution led to biphasic changes in the intensity of the transmitted light. Within 5 to 10 minutes of exposure to Ringer's solution, a general decrease in the light transmission across the long axis of the fiber was observed. Extended superfusion led to a local, apparent increase in light transmission corresponding to the formation of membrane blebs and globules. Images of disingerated globules focused above their equator showed bright halos with dark central zones. In electron micrographs, the single fibers showed uniform electron density. No significant inhomogeneities or precipitation of intracellular crystallins was observed in globules generated from fiber cells exposed to Ringer's solution; in addition, no high molecular weight protein aggregates were found in the globules.

CONCLUSIONS

Exposure to calcium alters the light-transmitting properties of isolated cortical fibers. The initial decrease in the average light transmittance of the fiber appears to be secondary to cell swelling and may relate to protein-based opacification. An apparent increase in light transmission through calcium-generated globules is likely because of the Becke line generated by a mismatch between the refractive index of the medium and the globule cytoplasm and accentuated by the transition from rod-shaped to spheroidal morphology.

目的

本研究的目的是检测在大鼠晶状体皮质单纤维崩解性球状体化过程中光透过率的变化。

方法

通过在含有10 mM HEPES、10 mM EDTA和280 mM蔗糖（pH 7.4，300至310 mOsm）的HEPES-EDTA-蔗糖（HES）溶液中用胰蛋白酶处理，从成年大鼠晶状体中分离出单皮质纤维。分离出的纤维用白色光源照射，通过电荷耦合器件相机收集透过纤维的光，并通过数字图像分析进行定量。在一些实验中，使用透射电子显微镜检查固定晶状体细胞的薄片。

结果

晶状体的酶解产生细长纤维，在林格氏液（含2 mM Ca2+）存在下，这些纤维发生崩解性球状体化。对悬浮在HES溶液中的细长纤维的光透过率测量显示，在纤维中心处透过率最高。将皮质纤维暴露于林格氏液会导致透射光强度发生双相变化。在暴露于林格氏液5至10分钟内，观察到沿纤维长轴的光透过率普遍下降。长时间灌注导致与膜泡和球状体形成相对应的局部光透过率明显增加。聚焦于赤道上方的崩解球状体图像显示出明亮的光晕和黑暗的中心区域。在电子显微镜照片中，单纤维显示出均匀的电子密度。在暴露于林格氏液的纤维细胞产生的球状体中未观察到明显的不均匀性或细胞内晶状体沉淀；此外，在球状体中未发现高分子量蛋白质聚集体。