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Y. Luo, S. Sinclair, W. Li; Microarray Analysis of Apoptosis-Related Genes Expressed by Retinal Microvascular Cells in Early Diabetic Rats . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4542.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To detect early expressed apoptosis-related genes by retinal microvascular (MV) cells in early diabetes. Methods: Sprague-Dawley male rates were induced to diabetes by intraperitoneal injection of streptozotocin. Hyperglycemia was monitored and the morphologic changes of retinal vessels were examined at the designed intervals. The rats were scarified at 3, 6, and 9 weeks after the induction of diabetes. In each experiment, 6 to 8 retinas were pooled and retinal microvessels were then purified for total RNA extraction. Nylon slides containing 100 apoptosis-related genes were hybridized to 32P-dCTP-labeled cDNAs that were synthesized using total RNAs. The phosphor screen scanned image was analyzed with GEArrayAnalyzer software. Results: The purity of enriched retinal microvessels was proved by interference microscopy. 2 to 5 µg total RNA were extracted from 6 to 8 retinal MV preparations. Of the 100 genes represented on the nylon slides, 11%, 18%, and 24% hybridized to the MV cDNA targets prepared from diabetic rats at the 3, 6 and 9-week intervals, respectively. The upregulation (>2-fold) of caspase 8 and NF-КB began at the 3-week interval, and the increase in expression of caspase 3 was detected at the 6- and 9-week intervals in replicate experiments. Conclusions: Enriched retinal MV preparation used in the present study has revealed specific molecular events of retinal MV cells undergoing apoptosis in early diabetes. Using slidemicroarrays containing candidate genes previously identified with apoptosis, the number of candidate genes detected in MV cells was increased with the duration of hyperglycemia. The increased expression of caspase 8 along with NF-КB at the earliest and caspase 3 at the later intervals suggest that at least Fas/APO-1/CD95 pathway and possible a Fas-independent pathway are involved in signaling mechanisms that lead to retinal MV cell apoptosis in early diabetes.
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