Location of exogenous α-crystallin in retinal ganglion cells
10.3760/cma.j.issn.2095-0160.2011.08.003
- VernacularTitle:外源性α晶状体蛋白作用于视网膜神经节细胞的定位研究
- Author:
Xing-li, WANG
;
Qiang, MA
;
Yu-xiao, ZENG
;
Jian-rong, HE
;
Yi, WANG
- Publication Type:Journal Article
- Keywords:
α-Crystallin;
Biotinylated;
Retinal ganglion cells
- From:
Chinese Journal of Experimental Ophthalmology
2011;29(8):681-684
- CountryChina
- Language:Chinese
-
Abstract:
Background There is no effective method to regenerate the optic nerve after injury. It has been recently reported that α-crystallin could promote the survive rate and axon regeneration of retinal ganglion cells (RGCs) effectively. However,the molecular mechanism is not clear. Objective This study was to identify the site of RGCs where the exogenous α-crystallin bind to. Methods RGCs was isolated from retinas of two 2-day-old Long Evans rats and primarily cultured. The positive rate of the RGCs was assessed by counting the number of positive cells for fluorescently-labeled thy1. 1 and cy3 under the fluorescence microscope. The biotinylated exogenous α-crystallin was evaluated by direct coloration and the activity of molecular chaperones was measured by insulin test.After identifying the success of biotinylation along with the activity of molecular chaperones,biotinylated α-crystallin was co-incubated with RGCs and the cells then were reacted to fluorescently labeled avidin for the observation of binding site of exogenous α-crystallin under the laser confocal microscope. Results RGCs of 94% were survived through primary culture. The coloration of biotinylated α-crystallin labeled by the direct coloration method was more intensive, and the value of A450 descended as the decrease of biotinylated α-crystallin concentration,indicating that the α-crystallin was biotinylated successfully. The activity of molecular chaperones of biotinylated α-crystallin was significantly strong but no significant change after being biotinylated after co-incubation of RGCs with biotinylated α-crystallin. Laser confocal microscope examination revealed that co-incubated RGCs with biotinylated α-crystallin showed the red fluorescence on membrane and axon of RGCs rather than cytoplasm and nucleus. The absent response was seen in the control group. Conclusion Exogenous α-crystallin can specifically combine with the membrane of RGCs to play the biological function,but its binding mode and mechanism need further study.
