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Objective To investigate the neuro-protective effect of bone marrow mesenchymal stem cells (BMSCs)transplantation in cerebral ischemia/reperfusion injury model. Methods The modified Zea-Longa suture method was adopted to establish rat middle cerebral artery occlusion(MCAO)/reperfusion injury model.Experimen-tal rats were randomly divided into blank group,model group,PBS group and BMSCs transplantation group. PBS group and BMSCs transplantation group were respectively received tail vein injection of 1 mL PBS or BMSCs(l × l06)6 h after the establishing of MCAO/reperfusion model. Neurological function was evaluated using the modified neurologic severity score(mNSS)scale at 12 h,1 d,3 d and 7 d after operation.Quantitative real-time PCR was used to detect the levels of miR-34a and miR-21 in ischemic brain tissue. Results(1)The mNSS scores in BM-SCs transplantation group showed no significant difference when compared with those in model group at 12 h and 1 d after operation(P > 0.05),whereas were significantly lower than those in model group at 3 d and 7 d(P <0.05).(2)Compared with model group,BMSCs transplantation group showed decreased level of miR-34a(P <0.01),while elevated level of miR-21 at different time points(P<0.01). Conclusions BMSCs transplantation can improve the impaired neurological function induced by cerebral ischemia/reperfusion injury,showing neuro-pro-tective effect.The mechanism may be associated with regulating the expressions of apoptosis-related microRNAs.
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Objective To investigate the possible mechanism of recombinant human erythropoietin (rhEPO) neuroprotection by studying the effect of rhEPO on expressions of matrix metalloproteinase-9 (MMP-9) and BCL-2 following focal cerebral ischemia-reperfusion in rats. Methods A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was induced by the intraluminal filament method, and intraperitoneal injection of rhEPO was used for intervention. Histopathological changes were observed by HE staining, and the expressions of MMP-9 and BCL-2 in the cerebral cortex of ischemic side were detected with immunohisto-chemistry. Results HE staining: At all time points, the numbers of surviving nerve cells were significantly higher in the rhEPO group, and their injury degree was significantly lower. MMP-9 immunohistochemistry staining: The positive cells were observed occasionally in the normal control group and the sham-operation group; the MMP-9 positive cells at the ischemic side of brain tissue in a normal saline control group began to appear at 6 hours after reperfusion, it reached the peak at 24 hours and began to decrease at 72 hours; the change trend of MMP-9 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly lower than that in the normal saline control group at the same time points (t were 12. 023 6, 12. 635 0, 12. 779 6, respectively, all P <0. 01). BCL-2 immunohistochemistry staining: No positive cells were found in the normal control group and sham-operation group. The numbers of BCL-2 positive cells reached the peak at the ischemic side of brain tissue in the normal saline control group at 6 hours after reperfusion, it reached the peak at 24 hours and further decreased at 72 hours; the change trend of BCL-2 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly higher than that in the normal saline control group at the same time points (t were 5. 763 1,8. 110 1, and 5. 798 7, respectively, all P <0. 01). Conclusions rhEPO may inhibit cortical neuronal apop-tosis at the ischemic side by inhibiting MMP-9 expression and up-regulating BCL-2 expression so as to play a neuroprotective effect.
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@#Objective To explore the protective effect of erythropoietin(EPO)on inflammatory injury induced by focal cerebral ischemia/reperfusion in rats.Methods 54 male Sprague-Dawley rats were randomly divided into 3 groups:sham operation group,normal saline control group and EPO group.The focal cerebral ischemia-reperfusion injury model was made by suture-occluded method.The content of interleukin-1 β(IL-1β)and the activity of myeloperoxidease(MPO)were measured by radioimmunoassay and chromatoptometry at 6 h,24 h and 48 h reperfusion following ischemia 2 h.The scores of behavior obstacle in rats were assessed at 24 h.Results Compared with the normal saline control group,EPO group got better score of behavior obstacle(all P<0.05).The content of IL-1β and the activity of MPO in brain in the EPO group were significantly lower than those in the normal saline control group(all P<0.01).Compared with two reperfusion groups,the sham operation group had no obvious abnormal change in each measurement item.Conclusion EPO can reduce the content of IL-1β and inhibit the infiltration of leukoeyte,which can provide protective effect on cerebral ischemic-repefusion injury in rats.
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The studies of animal models have shown that the expression of matrix metalloprotease (MMP) is abnormal during cerebral ischemia/reperfusion,which indicating that it is associated with cerebral ischemia/reperfusion injury,especially MMP-2,MMP-3 and MMP-9 play the important roles in the pathogenesis of acute ischemic cerebrovascular disease.Understanding of the roles and expressions of MMP,the factors affecting its expression,and the studies and application of the related antibodies in cerebral ischemic/reperfusion injury may provide a new alternative for the early diagnosis,prevention and treatment of cerebral infarction.
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AIM: To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6?His tag, and to determine the affinity constant of the purified recombinant product.METHODS: Solubilizing in buffers containing urea or guanidine hydrochloride (GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni 2+ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA. RESULTS: The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached (61.08?1 45)%. The affinity constant of the polished scFv was confirmed to be(2.30?0.32) ?10 7 L/mol. CONCLUSION: The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro . The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.
