【摘要】：目的:通過(guò)建立五步蛇毒大鼠中毒模型,觀察五步蛇毒對(duì)大鼠SVZ處Nestin基因及蛋白表達(dá)的影響,并探討HBO聯(lián)合抗毒血清干預(yù)對(duì)其的作用。方法:將96只健康成年雄性SD大鼠,隨機(jī)分為4組:對(duì)照組、蛇毒組、血清組、HBO+血清組,每組24只。經(jīng)尾靜脈注射五步蛇毒建立中毒模型,血清組于造模成功后立即尾靜脈注射抗毒血清,HBO+血清組于注射抗毒血清后0 h、4 h、11 h、23 h予以HBO治療一次,每個(gè)時(shí)間點(diǎn)各6只,對(duì)照組不予任何處理。上述各組中的一部分大鼠分別于HBO治療結(jié)束后的相應(yīng)時(shí)間點(diǎn)(即注射抗毒血清后1 h、5 h、12 h、24h)用生理鹽水及多聚甲醛經(jīng)左心室灌注固定腦組織,另一部分大鼠斷頭后立即冰上分離SVZ處組織,速凍備用,HE染色觀察腦組織病理變化,采用IHC、RT-PCR、Western-blot等檢測(cè)各組SVZ處Nestin m RNA及蛋白的表達(dá)變化。結(jié)果:1.腦組織病理變化:與對(duì)照組比較,蛇毒組SVZ處出現(xiàn)明顯組織水腫,細(xì)胞數(shù)目減少,排列紊亂;經(jīng)抗毒血清和HBO干預(yù)后SVZ處組織水腫明顯減輕,細(xì)胞數(shù)增多,排列較規(guī)則;HBO聯(lián)合干預(yù)組病理變化改善更突出,且以注毒后5 h內(nèi)最明顯。2.SVZ處Nestin基因及蛋白表達(dá)變化:Nestin蛋白在顱內(nèi)表達(dá)于NSCs胞漿。IHC結(jié)果提示對(duì)照組未見Nestin陽(yáng)性細(xì)胞,蛇毒組僅有少許Nestin陽(yáng)性表達(dá)細(xì)胞,抗毒血清和HBO干預(yù)后Nestin陽(yáng)性細(xì)胞明顯增多,著色加深(P0.05)。相對(duì)定量結(jié)果顯示:與對(duì)照組比較,蛇毒組各時(shí)間點(diǎn)SVZ處Nestin m RNA和蛋白的表達(dá)量均明顯升高(P0.05);與蛇毒組相比,血清組、HBO+血清組Nestin m RNA和蛋白的表達(dá)量又進(jìn)一步升高,注毒后1 h、5 h時(shí)差異顯著(P0.05),而在HBO+血清組其表達(dá)水平又顯著高于血清組(P0.05)。結(jié)論:1.五步蛇毒可對(duì)腦組織造成神經(jīng)毒性損傷;2.HBO聯(lián)合抗毒血清干預(yù)可對(duì)五步蛇毒中毒后腦損傷發(fā)揮保護(hù)作用,可能與SVZ處Nestin蛋白的上調(diào)有關(guān);3.HBO聯(lián)合抗毒血清早期干預(yù)的腦保護(hù)效果優(yōu)于單獨(dú)使用抗毒血清,尤其在五步蛇毒中毒后5 h內(nèi)作用最顯著。
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R646

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