Résumé
Objective To study the changes of SUR1-regulated NCCa-ATP channels in core area during focal cerebral ischemia and reperfusion in rats, and to explore the therapeutic window of focal cerebral ischemia. Methods Adult male Wistar rats were rendered to undergo 120 minutes of the left middle cerebral artery occlusion (MCAO) by the intraluminal thread technique before reperfusion. The core area of the left hemisphere cortex ischemia/reperfusion at different times (reperfusion:R3 h, 6 h, 8 h, 12 h and 24 h) was taken to be tested the level of mRNA and protein expression of sulfonylurea receptor 1 (SUR1) using RT-PCR and Western-blot techniques. SUR1 of the ischemic brain microvascular endothelial cells were observed by immunofluorescence double staining at the peak expressing time point of SUR1. Results We found the up-regulation of SUR1 mRNA and SUR1 protein in ischemic infarcts tissues for R3 h, 6 h, 8 h and 12 h, peaked at R8 h, compared with the sham-operation group ( <0.05) . However, SUR1 expression increased was observed at R12 h by double immunofluorescence in microvascular endothelium. Conclusion SUR1-regulated NCCa-ATP channel may take part in cerebral ischemic damage during focal cerebral ischemia and reperfusion. The expressions of SUR1 mRNA and SUR1 protein were up-regulated in ischemic infarcts tissues. The data suggested that the best time to apply SUR1 inhibitor is within 8-12 hours after ischemia and reperfusion.
Résumé
<p><b>OBJECTIVE</b>To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population.</p><p><b>METHODS</b>Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites.</p><p><b>RESULTS</b>PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study.</p><p><b>CONCLUSION</b>The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.</p>