【摘要】：目的:應(yīng)用抗體阻斷法治療人類內(nèi)毒素血癥已有多年歷史,但由于抗內(nèi)毒素血清制備成本高,尚未在獸醫(yī)臨床上使用�；诖�,本實(shí)驗(yàn)通過(guò)制備抗大腸桿菌內(nèi)毒素血清中特異性IgG抗體,探究IgG抗體對(duì)內(nèi)毒素血癥的療效,為臨床治療內(nèi)毒素血癥提供實(shí)驗(yàn)和理論依據(jù)。方法:采集病犬糞便,細(xì)菌培養(yǎng)分離并鑒定為大腸桿菌;三氯醋酸法提取的大腸桿菌內(nèi)毒素經(jīng)鱟試劑活性鑒定后,免疫實(shí)驗(yàn)犬2 w,待效價(jià)達(dá)1:64后,加強(qiáng)免疫1 w,頭靜脈采血,血清分離;將血清樣品經(jīng)辛酸-硫酸銨法純化后,所得上清液即為純化的IgG抗體液,置于-20℃凍存。昆明小鼠36只,隨機(jī)分為對(duì)照組、內(nèi)毒素血癥組、血清治療組,每組12只。對(duì)照組,給予等量生理鹽水;內(nèi)毒素血癥組,腹腔注射10 mg/kg的LPS;血清治療組,腹腔注射10 mg/kg的LPS后,立即皮下注射抗大腸桿菌內(nèi)毒素特異性IgG抗體,連續(xù)用藥1 w,1次/d。電子體溫計(jì)測(cè)量每組用藥后小鼠體溫,2次/d,連續(xù)1 w;比較用藥后各組體溫變化。血細(xì)胞分析儀測(cè)定用藥后1、3、7 d血常規(guī)參數(shù)指標(biāo);比較用藥后各組血常規(guī)參數(shù)變化。ELISA法測(cè)定每組血清中腫瘤壞死因子-ɑ和白介素-6的含量;比較用藥后各組TNF-α和IL-6含量變化。實(shí)驗(yàn)結(jié)束后,犧牲動(dòng)物,開(kāi)腹觀察肝臟和腎臟變化;取出肝臟和腎臟,中性福爾馬林溶液固定,制作病理切片,HE染色,顯微鏡下觀察肝臟中肝小葉、肝細(xì)胞索的變化及炎性細(xì)胞浸潤(rùn)程度;腎臟中腎小管管型、腎小管上皮細(xì)胞的變化及炎性細(xì)胞浸潤(rùn)程度。結(jié)果:1.體溫變化:與對(duì)照組比較,第1~7 d陽(yáng)性對(duì)照組體溫極顯著升高(P0.01)。與陽(yáng)性對(duì)照組比較,第1~7 d血清治療組溫度極顯著降低(P0.01)。2.血常規(guī)變化:與對(duì)照組比較,第1、3、7 d陽(yáng)性對(duì)照組WBC、LYM均極顯著降低(P0.01)。與陽(yáng)性對(duì)照組比較,第1、3、7 d血清治療組WBC、LYM均極顯著升高(P0.01)。與對(duì)照組比較,第1、3、7 d陽(yáng)性對(duì)照組GRAN極顯著升高(P0.01)。與陽(yáng)性對(duì)照組比較,第1、3、7 d血清治療組GRAN極顯著降低(P0.01)。3.TNF-α和IL-6:對(duì)照組TNF-α和IL-6的含量為332.7±10.2 ng/L和114.5±4.06 ng/L,陽(yáng)性對(duì)照組為460.6±11.8 ng/L和189.5±20.9 ng/L,血清治療組為367.6±7.6 ng/L和128.9±7.8 ng/L,血清治療組TNF-α和IL-6含量均較陽(yáng)性對(duì)照組極顯著降低(P0.01)。4.病理變化:各組病理變化情況與空白組相比有明顯好轉(zhuǎn)的趨勢(shì),肝細(xì)胞索排列規(guī)則、炎性細(xì)胞浸潤(rùn)減少、部分細(xì)胞核體積恢復(fù)正常;腎小管上皮細(xì)胞排列整齊,間質(zhì)中炎性細(xì)胞浸潤(rùn)明顯減少。結(jié)論:抗內(nèi)毒素血清能夠拮抗血液中內(nèi)毒素,降低炎性細(xì)胞數(shù),減輕炎性細(xì)胞浸潤(rùn)程度,減少腫瘤壞死因子-α和白介素-6的表達(dá)量,從而緩解內(nèi)毒素對(duì)肝臟和腎臟的損傷。
[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，

本文編號(hào)：2461007