犬源抗内毒素血清的制备及其疗效初探
发布时间：2019-04-19 14:05
【摘要】：目的:应用抗体阻断法治疗人类内毒素血症已有多年历史,但由于抗内毒素血清制备成本高,尚未在兽医临床上使用。基于此,本实验通过制备抗大肠杆菌内毒素血清中特异性IgG抗体,探究IgG抗体对内毒素血症的疗效,为临床治疗内毒素血症提供实验和理论依据。方法:采集病犬粪便,细菌培养分离并鉴定为大肠杆菌;三氯醋酸法提取的大肠杆菌内毒素经鲎试剂活性鉴定后,免疫实验犬2 w,待效价达1:64后,加强免疫1 w,头静脉采血,血清分离;将血清样品经辛酸-硫酸铵法纯化后,所得上清液即为纯化的IgG抗体液,置于-20℃冻存。昆明小鼠36只,随机分为对照组、内毒素血症组、血清治疗组,每组12只。对照组,给予等量生理盐水;内毒素血症组,腹腔注射10 mg/kg的LPS;血清治疗组,腹腔注射10 mg/kg的LPS后,立即皮下注射抗大肠杆菌内毒素特异性IgG抗体,连续用药1 w,1次/d。电子体温计测量每组用药后小鼠体温,2次/d,连续1 w;比较用药后各组体温变化。血细胞分析仪测定用药后1、3、7 d血常规参数指标;比较用药后各组血常规参数变化。ELISA法测定每组血清中肿瘤坏死因子-ɑ和白介素-6的含量;比较用药后各组TNF-α和IL-6含量变化。实验结束后,牺牲动物,开腹观察肝脏和肾脏变化;取出肝脏和肾脏,中性福尔马林溶液固定,制作病理切片,HE染色,显微镜下观察肝脏中肝小叶、肝细胞索的变化及炎性细胞浸润程度;肾脏中肾小管管型、肾小管上皮细胞的变化及炎性细胞浸润程度。结果:1.体温变化:与对照组比较,第1~7 d阳性对照组体温极显著升高(P0.01)。与阳性对照组比较,第1~7 d血清治疗组温度极显著降低(P0.01)。2.血常规变化:与对照组比较,第1、3、7 d阳性对照组WBC、LYM均极显著降低(P0.01)。与阳性对照组比较,第1、3、7 d血清治疗组WBC、LYM均极显著升高(P0.01)。与对照组比较,第1、3、7 d阳性对照组GRAN极显著升高(P0.01)。与阳性对照组比较,第1、3、7 d血清治疗组GRAN极显著降低(P0.01)。3.TNF-α和IL-6:对照组TNF-α和IL-6的含量为332.7±10.2 ng/L和114.5±4.06 ng/L,阳性对照组为460.6±11.8 ng/L和189.5±20.9 ng/L,血清治疗组为367.6±7.6 ng/L和128.9±7.8 ng/L,血清治疗组TNF-α和IL-6含量均较阳性对照组极显著降低(P0.01)。4.病理变化:各组病理变化情况与空白组相比有明显好转的趋势,肝细胞索排列规则、炎性细胞浸润减少、部分细胞核体积恢复正常;肾小管上皮细胞排列整齐,间质中炎性细胞浸润明显减少。结论:抗内毒素血清能够拮抗血液中内毒素,降低炎性细胞数,减轻炎性细胞浸润程度,减少肿瘤坏死因子-α和白介素-6的表达量,从而缓解内毒素对肝脏和肾脏的损伤。
[Abstract]:Aim: antibody blocking has been used to treat human endotoxemia for many years, but it has not been used in veterinary clinic because of the high cost of preparation of anti-endotoxin serum. In order to provide experimental and theoretical basis for clinical treatment of endotoxemia, we prepared anti-Escherichia coli endotoxin serum specific IgG antibody to explore the therapeutic effect of IgG antibody on endotoxemia. Methods: the feces of sick dogs were collected and isolated by bacterial culture and identified as Escherichia coli. The endotoxin extracted from Escherichia coli by trichloroacetic acid method was identified by Limulus reagent. The dogs were immunized for 2 w and the titer was up to 1:64. After 1 w immunization, the blood was collected from the head vein and the serum was separated. After the serum samples were purified by octanoic acid-ammonium sulfate method, the supernatant was purified IgG antibody solution and stored at-20 鈩，
本文编号：2461007
[Abstract]:Aim: antibody blocking has been used to treat human endotoxemia for many years, but it has not been used in veterinary clinic because of the high cost of preparation of anti-endotoxin serum. In order to provide experimental and theoretical basis for clinical treatment of endotoxemia, we prepared anti-Escherichia coli endotoxin serum specific IgG antibody to explore the therapeutic effect of IgG antibody on endotoxemia. Methods: the feces of sick dogs were collected and isolated by bacterial culture and identified as Escherichia coli. The endotoxin extracted from Escherichia coli by trichloroacetic acid method was identified by Limulus reagent. The dogs were immunized for 2 w and the titer was up to 1:64. After 1 w immunization, the blood was collected from the head vein and the serum was separated. After the serum samples were purified by octanoic acid-ammonium sulfate method, the supernatant was purified IgG antibody solution and stored at-20 鈩，
本文编号：2461007
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