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Cerebral ischemic stroke (ischemic stroke, IS) in the world have a very high morbidity, mortality and neurological morbidity. The IS incidence rate is gradually increased with the advent of an aging society, still further increase the hazard to human health. With CT, MRI and other imaging techniques, you can check out the majority of IS, but early in the disease or even in some cases, be able to make timely diagnosis is still difficult. And found that cerebral ischemic injury specificity and sensitivity of molecular markers, to help create a new early molecular diagnostic methods. In June 2006, Ladenson research team of the University of Washington reported that the use of gene array technology, bioinformatics analysis of screening combined with further confirmed by Western blot strategy to filter out the the brain high abundance, high-specific expression of protein: cone-like protein - 1 (visinin-like protein 1, VILIP-1); in IS patients and animal models in vivo, they also detected VILIP-1 upregulation. Therefore, VILIP-1 is expected to become important molecular markers of ischemic brain injury. Vsnl1 VILIP-1 protein coding genes, the total length of 576 bp cDNA. GenBank database data show that in humans, mice and rats, vsnl1 sequence is basically the same, and in the basal ganglia, forebrain, cerebellum and brain brain tissue and the vast majority of nerve cells in the brain are highly expressed. VILIP-1 under the calcineurin sensing protein (NCS) a member of the family VILIP subfamily constitutively expressed in healthy brain tissue characteristics have been initially elucidated, but as an IS closely related molecules in animal models even expression characteristics of IS patients remains unclear. The this study vsnl1 Cloning and prokaryotic expression and the establishment of a suture method the IS rat model, further of gene transcription vsnl1 level changes were studied. , Vsnl1 Cloning and prokaryotic expression First, optimized PrimerSelect software design primer sequences. Then the whole brain from healthy BALB / c mice, with BIOZOL reagent total RNA was extracted and purified. RNase-free DNase I digestion to remove residual DNA, the line of the reverse transcription reaction cDNA, the latter template for PCR reaction amplification vsnl1. The purpose PCR fragments were ligated to pMD18-T, after restriction enzyme digestion and sequencing, the sequence submitted to GenBank parallel BLAST alignment analysis. The subcloned technology to construct the prokaryotic expression plasmid pROEX-v containing 6xHis tag and transformed into E. coli DH5a line IPTG induction and Ni-NTA column purification. The results show that: (1) two-step RT-PCR amplification product of 576 bp, and vsnl1 gene expects the same size; (2) Sequencing and BLAST alignment analysis suggests the presence of the NT205 (TC) and NT314 (TA ) two mutant sites, and the published vsnl1 homology to 99%, the gene sequence has been submitted to GenBank for accession number EU373446; (3) successful to build pROEX-v induced expression product was confirmed by SDS-PAGE, of approximately 22 kDa at the destination protein bands, consistent with the expected molecular size; (4) SDS-PAGE analysis of the sample line was purified on Ni-NTA column, the results also show that the purpose of purified protein bands at approximately 22 kDa, but The yield is slightly lower. Second, the ischemic stroke rat model the Preparation and vsnl1 Transcription First Reference reported intraluminal filament model approach, the establishment of the right side of the rat brain artery occlusion (middlecerebral arterial occlusion, MCAO) model, permanent ( pMCAO) and transient (tMCAO) obstruction of animal models 11, 10 did not undergo MCA occlusion rat control. Neurological score, and brain biopsy for histopathological identification of animals. Secondly, after the perfusion of the heart, the brain extract infarction of the brain specimens (right) side and infarct (left) side of the total RNA, cDNA was obtained by reverse transcription complete digestion to remove residual DNA. Once again, the line of semi-quantitative PCR, and grayscale scanning using Quantity One software line, vsnl1 integral ratio of the optical density of the beta-actin as a semi-quantitative indicators for comparative analysis. Finally, by DNAMAN software the PrimerSelect software to optimize the design of quantitative PCR primers, quantitative PCR, a group of brain specimens infarct side and not the infarct side of cDNA as a template, further analysis of the quantitative detection of vsnl1 transcriptional level line. The results show that: (1) neurological score infarction after 24 h MCAO rats showed significant symptoms of neurological deficit (pMCAO, 2.64 ± 1.03; tMCAO, 2.27 ± 0.90; control, 0), pMCAO and tMCAO group and control group were statistically significant difference (P <0.05), while no significant difference (P> 0.05) between pMCAO and tMCAO group. The results also suggest that the tMCAO group than the pMCAO group seems to have a longer survival time, suggesting the early reperfusion beneficial to extend the IS-touch survival time. (2) TTC staining showed that pMCAO and tMCAO rat model of brain tissue in the right MCA territory, including parts of the cerebral cortex, striatum and hippocampus are seen clearly in the infarct; normal rats and MCAO rats the left side of the brain for the uniform red dye area, the infarct. The HE staining microscope results show that the the MCAO rat infarct brain tissue have more infarct and micro-infarct, it is also visible cell edema, degeneration, necrosis and other pathological changes, while the control normal. Still found that the the pMCAO rats than tMCAO infarct more severe manifestations of infarct size and number of lesions. (3) semi-quantitative PCR results showed that both sides the brain tissue vsnl1 transcriptional level similar to the control group, the left and right. In the pMCAO and tMCAO group, ischemic brain tissue the vsnl1 transcriptional level lower than the non-ischemic side. (4) quantitative PCR, vsnl1 relative transcript levels (vsnl1 mRNA / β-actin mRNA), in pMCAO non-ischemic side as 0.040 ± 0.007, the ischemic side 0.023 ± 0.002; in tMCAO non-ischemic side 0.049 ± 0.011, ischemic side was 0.030 ± 0.002. Tips, the ischemic side vsnl1 transcription level is below the non-ischemic side, although no statistically significant. In the control group, the left and right sides vsnl1 transcription level is substantially the same. We speculate that disruption of calcium homeostasis may be the MCAO the brain tissue vsnl1 transcription down one of the important mechanisms. But, in view of the complex role of VILIP-1 has a pro-apoptotic role and a variety of signaling cascades regulating functions, the detailed mechanism remains to be confirmed by further studies in the future.
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