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Dan Hu, Li-Juan Sun; Effect of hypoxia treatment by CoCl2 on expression of glutamate/aspartate transporters (GLAST) and unfolded protein reaction (UPR) related factors in retinal Müller cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):410.
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Glaucoma and other optic nerve diseases are some of the major causes of blindness which are characterized by progressive death of the retinal ganglion cells (RGCs) and decreasing number of retinal nerve fiber. Müller cells, the principal glial cells of the retina, play an important role in neuroprotection against glutamate-induced cytotoxicity in RGCs. Recent studies have shown that UPR may be one mainly pathological mechanism of the progressive death of RGCs and the decreasing number of retinal nerve fiber. The present study evaluated the effect of hypoxia on expression of the UPR related factors X-box binding protein 1 (XBP1) and C/EBP homologous protein (CHOP) in Müller cells, thus clarifying the relationship between UPR and GLAST.
Müller cells were isolated from the retinas of rats on postnatal day 3~7 by using an enzymatic digestion-mechanical trituration technique. Optical microscopy and immunocytochemical method were used to evaluate cells. Cultured retinal Müller cells were exposed to either 0.2mM CoCl2 or 2mM DTT for 0, 3, 6, 12, 24, 48, and 72 in vitro, respectively. We used Real Time PCR to measure sequential changes of the XBP1, CHOP and GLAST mRNA expression. Levels of XBP1 and GLAST proteins were monitored by immunoblot analysis.
At least 90% cultured cells showed positive reaction for GLAST and glutamine synthetase (GS) with an immunocytochemical assay. The positive rate of GLAST expression was 84.66±3.51% determined by flow cytometry. The expression levels of XBP1, CHOP and GLAST were all ascended at the early stage, reached the summit at 24 hours after hypoxia induced by CoCl2 and then dropped sharply after 72h, which was consistent with the results of DTT treatment. The protein expression of XBP1, CHOP and GLAST were similar to the transcription level.
These results suggest that UPR exists in the Müller cells after hypoxia, and its related factors XBP1, CHOP expression are equal to GLAST. Our study should facilitate further study of the role of URP in eye diseases and identification of the potential therapeutic targets.
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