高压氧辅助抗毒血清干预对五步蛇毒中毒大鼠SVZ的影响

发布时间：2018-08-28 13:35

【摘要】：目的:通过建立五步蛇毒大鼠中毒模型,观察五步蛇毒对大鼠SVZ处Nestin基因及蛋白表达的影响,并探讨HBO联合抗毒血清干预对其的作用。方法:将96只健康成年雄性SD大鼠,随机分为4组:对照组、蛇毒组、血清组、HBO+血清组,每组24只。经尾静脉注射五步蛇毒建立中毒模型,血清组于造模成功后立即尾静脉注射抗毒血清,HBO+血清组于注射抗毒血清后0 h、4 h、11 h、23 h予以HBO治疗一次,每个时间点各6只,对照组不予任何处理。上述各组中的一部分大鼠分别于HBO治疗结束后的相应时间点(即注射抗毒血清后1 h、5 h、12 h、24h)用生理盐水及多聚甲醛经左心室灌注固定脑组织,另一部分大鼠断头后立即冰上分离SVZ处组织,速冻备用,HE染色观察脑组织病理变化,采用IHC、RT-PCR、Western-blot等检测各组SVZ处Nestin m RNA及蛋白的表达变化。结果:1.脑组织病理变化:与对照组比较,蛇毒组SVZ处出现明显组织水肿,细胞数目减少,排列紊乱;经抗毒血清和HBO干预后SVZ处组织水肿明显减轻,细胞数增多,排列较规则;HBO联合干预组病理变化改善更突出,且以注毒后5 h内最明显。2.SVZ处Nestin基因及蛋白表达变化:Nestin蛋白在颅内表达于NSCs胞浆。IHC结果提示对照组未见Nestin阳性细胞,蛇毒组仅有少许Nestin阳性表达细胞,抗毒血清和HBO干预后Nestin阳性细胞明显增多,着色加深(P0.05)。相对定量结果显示:与对照组比较,蛇毒组各时间点SVZ处Nestin m RNA和蛋白的表达量均明显升高(P0.05);与蛇毒组相比,血清组、HBO+血清组Nestin m RNA和蛋白的表达量又进一步升高,注毒后1 h、5 h时差异显著(P0.05),而在HBO+血清组其表达水平又显著高于血清组(P0.05)。结论:1.五步蛇毒可对脑组织造成神经毒性损伤;2.HBO联合抗毒血清干预可对五步蛇毒中毒后脑损伤发挥保护作用,可能与SVZ处Nestin蛋白的上调有关;3.HBO联合抗毒血清早期干预的脑保护效果优于单独使用抗毒血清,尤其在五步蛇毒中毒后5 h内作用最显著。
[Abstract]:Aim: to investigate the effect of HBO combined with antivenom on the expression of Nestin gene and protein in SVZ rats by establishing a rat poisoning model of APV venom. Methods: 96 healthy adult male SD rats were randomly divided into 4 groups: control group, snake venom group and serum group with 24 rats in each group. The poisoning model was established by injecting the venom of Viper venom via tail vein. The serum group was treated with HBO at 4 h and 11 h and 23 h after injection of antitoxic serum immediately after the successful establishment of the model, with 6 rats in each time point. No treatment was given in the control group. Some of the rats in each group were perfused with normal saline and paraformaldehyde at the corresponding time points after HBO treatment (i.e. 1 h, 5 h, 12 h and 24 h after injection of antitoxic serum) to immobilize brain tissue through left ventricle. In another part of the rats, the tissues of SVZ were isolated on ice immediately after decapitation, and the pathological changes of brain tissue were observed by HE staining, and the expression of Nestin m RNA and protein in SVZ were detected by IHC,RT-PCR,Western-blot and so on. The result is 1: 1. The pathological changes of brain tissue: compared with the control group, there were obvious tissue edema, decreased cell number and disordered arrangement in SVZ in snake venom group, tissue edema and cell number increased in SVZ after intervention of antitoxic serum and HBO. The changes of Nestin gene and protein expression at SVZ within 5 hours after injection showed that Nestin positive cells were not found in the control group, and the expression changes of Nestin gene and protein in the brain were found in the NSCs cytoplasm. The results showed that there were no Nestin positive cells in the control group, and the results showed that there were no Nestin positive cells in the control group, and the results showed that there were no Nestin positive cells in the control group. There were only a few Nestin positive cells in snake venom group, and Nestin positive cells increased significantly after intervention of antitoxic serum and HBO (P0.05). The relative quantitative results showed that the expression of Nestin m RNA and protein at SVZ in snake venom group was significantly higher than that in control group (P0.05), and the expression of Nestin m RNA and protein in serum group was higher than that in snake venom group. There was significant difference at 5 h after injection (P0.05), and the expression level in HBO serum group was significantly higher than that in serum group (P0.05). Conclusion 1. (2) HBO combined with antitoxic serum intervention can protect the brain injury after the poisoning of the snake venom. It may be related to the upregulation of Nestin protein in SVZ. 3. The protective effect of SVZ combined with antitoxic serum in early intervention was better than that of antivenom alone, especially within 5 hours after the poisoning of APV venom.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R646

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