| AIMGap junction is composed of connexin hexamer ,existing in adjacent cells. It is rapid channels for inorganic ion, micro-molecule and water-soluble substance and metabolites, and providing electrochemical cell communication. Abnormal changes of intercellular communication mediated by gap junction is a important pathogenic factor of cataract, which is paid attention to gradually these years. Epidemiological evidence indicates that, long-term ultraviolet radiation, especially the wavelength in 320nm-400nm, also called near ultraviolet, is absorbed buy crystalline lens mainly, so near ultraviolet radiation is considered to have close relation to cataract. The mechanism about cataract is not clear yet, and it is verified by some scholars that near ultraviolet, microwave irradiation et al will lead to damage in Rabbit Lens Epithelium cultured in Vitro and decrease of intercellular communication. In this experiment, near ultraviolet and Staurosporine is regarded as experimental factor. The propose is to study protective effect of intercellular communication induced by Staurosporine in Rabbit Lens Epithelium cultured in Vitro and the mechanism. In this experiment, near ultraviolet and Staurosporine is regarded as experimental factor. We also hope to make it clear whether near ultraviolet can activate the protein kinase c and result in cataract, and discover a new research direction of cataract medication.METHOD1. Primary cell culture of Rabbit Lens Epithelium and experimental classification , healthy and mature rabbits without eye disease are sacrificed by air embolism, The eyeballs were enucleated and disinfected. Anterior capsule is intercepted and divided into few pieces, digest by type I collagenase, then inoculated in the culture bottle. Culturing solution is changed first time 10-12 hour later , then Culturing solution is changed every two days. During cultured in vitro, Epithelium showed adherent polar growth and continuous proliferation which covered the most bottom of the flask after 7-10 days and could be subcultured. Second or Third generation of Lens Epitheliums are applicated in the study. Appropriate Staurosporine drug concentration is selected in the pre-experiment, radiation energy of near ultraviolet has been defined, then experimental group , normal group and control group is designed.2. Assessment of intercellular communication in Rabbit Lens Epithelium: The fluore-scence- recovery-after-photobleaching ( FRAP ) method was used to determine the Gap Junction Intercellular communication of experimental group ,normal group and control group respetively.3. Detection of the expression and function orientation of Cx43, Western blot techniques is employed to detect the levels of Cx43 and the indirect immunofluorescence histochemistry method is applied to analyze the localization of Cx43 in the Rabbit Lens EpitheliumRESULT1. Appropriate drug concentration of Staurosporine is 10-7mol/L , we tried 5 concentration of Staurosporine in the pre-experiment, then using discrepancy microscope to observe the cell and using trypan blue dye exclusion test to find the ratio of cell trauma. We find that high-concentration Staurosporine will lead to the rapid death of lens epithelium, and decreaseing the concentration will lead to lower ratio of cell trauma. while the concentration decreased to 10-7mol/L, there is no significant difference in the ratio of cell trauma between the experimental group and normal group.2. Assessment of intercellular communication in Rabbit Lens Epithelium: there is significant difference of the fluorescence recovery rate in the experimental group, normal group and control group, the fluorescence recovery rate of experimental group is higher than that of control group, and lower than that of normal group. 3.Western-blot results : There is statistical significance in the expression of Cx43 in the experimental group, normal group and control group, and the expression of Cx43 of experimental group is higher than that of control group, and lower than that of normal group.4. The localization of Cx43 in the Rabbit Lens Epithelium, near ultraviolet radiation can lead to internalization of Cx43. It means that the Cx43 expressed in the cell membrane has reduced, and expression of these Cx43 transfers into cytoplasm and cell nuclear. The internalization of Cx43 in experimental group is higher than that of control group, and lower than that of normal group.CONCLUSIONNear ultraviolet radiation lead to change of cell morphology, damage of intercellular communication, reduced expression of Cx43 and internalization of Cx43, and Staurosporine can protect the intercellular communication in Rabbit Lens Epithelium cultured in Vitro, and find near ultraviolet can activate the protein kinase c and result in cataract.
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