兔豆状囊尾蚴Ⅰ型胱抑素对脂多糖诱导小鼠脓毒症防治机制的研究  

Studies on the effect of Cysticercus pisiformis type Ⅰ cystatin on lipopolysaccharide-induced sepsis in mice

作　　者：车亮 王由森 石正发 李甲 羊倩倩 肖琴 孙晓林[1] CHE Liang;WANG You-sen;SHI Zheng-fa;LI Jia;YANG Qian-qian;XIAO Qin;SUN Xiao-lin(Parasite Laboratory,College of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Bio-pharmaceutical Factory of Zhongmu Industry Co.,Ltd.,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院寄生虫实验室,甘肃兰州730070 [2]中牧实业股份有限公司兰州生物药厂,甘肃兰州730070

出　　处：《中国预防兽医学报》2024年第8期837-846,共10页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(32160843)。

摘　　要：为深入探究兔豆状囊尾蚴半胱氨酸蛋白酶抑制剂(CPI)超家族成员I型胱抑素(Stefin)对脂多糖(LPS)诱导的小鼠脓毒症的治疗效果,本研究将重组菌pGEX-4T-CpStefin/BL21经IPTG诱导表达重组Stefin蛋白(r-Stefin),采用GST试剂盒纯化上述蛋白,并去除内毒素后,采用SDS-PAGE检测r-Stefin的纯化效果。结果显示,r-Stefin在大肠杆菌中获得了表达且纯化效果较好。将雄性BALB/c小鼠随机分为6组,每组16只,分别为A组(生理盐水100μL/只,阴性对照组)、B组:GST蛋白对照组(25μg/只)、C组:r-Stefin组(25μg/只)、D组:脓毒症组(0.2 mg/100μL LPS/只)、E组:GST蛋白干预组(0.2 mg/100μL/只LPS+GST蛋白25μg/只)、F组:r-Stefin干预组(0.2 mg/100μL LPS/只+r-Stefin 25μg/只)。经腹腔注射后观察各组小鼠的临床症状、绘制各组小鼠在LPS诱导后不同时间的生存率曲线。在LPS诱导后12 h和24 h对各组小鼠采血,采用ELISA检测各组小鼠血清中各炎性细胞因子的含量,通过全自动生化仪测定各组小鼠血清中肝、肾功能指标。结果显示,D、E组小鼠经LPS诱导后很快出现被毛发凌乱、活动量减少、精神沉郁等症状。F组小鼠上述症状在一定程度上得到改善。A、B、C组小鼠均健活。生存率曲线显示,D组小鼠在36 h内全部死亡,E组小鼠在48 h~60 h时的生存率由75%快速降至72 h时的37.5%,F组小鼠的生存率在72 h时达75%。A~C组小鼠生存率均达100%。ELISA结果显示,LPS诱导12 h时,D组、E组和F组小鼠血清中促炎细胞因子(IL-6、TNF-α和TNF-β)及抑炎细胞因子(IL-10)的含量与A组相比均极显著升高;D组小鼠血清中IL-10含量显著低于F组。TNF-α及TNF-β含量分别极显著和显著高于F组。LPS诱导24 h时,D组小鼠血清中IL-6、TNF-α和TNF-β含量极显著高于F组,IL-10含量极显著低于F组。经LPS诱导后12 h,D组、E组、F组小鼠血清中肝脏指标ALB的含量与A组相比均极显著下降(P<0.001),ALT含量均极显著升高。DIn order to deeply investigate the therapeutic effect of Cysticercus pisiformis type I cystatin(Stefin),a member of the cysteine protease inhibitor(CPI)superfamily,on lipopolysaccharide(LPS)-induced sepsis in mice.In this study,recombinant bacterium pGEX-4T-CpStefin/BL21 was induced by IPTG to express recombinant Stefin protein(r-Stefin),and the protein were purified by using GST kit.The expression and purification of r-Stefin was detected by SDS-PAGE after removalendotoxin.The results showed that r-Stefin was expressed in E.coli.There was one band at 36ku(r-Stefin),indicating that r-Stefin had a good purification effect.Male BALB/c mice were randomly divided into 6 groups with 16 mice in each group,namely,Group A(100μL of saline injection per mouse,negative control group),Group B:GST protein control group(25μg of GST protein per mouse),Group C:r-Stefin group(25μg of r-Stefin protein per mouse),Group D:sepsis group[100μL of LPS(containing 0.2mg LPS,hereinafter)],Group E:GST protein intervention group(100μL LPS and 25μg of GST protein per mouse),and Group F:r-Stefin intervention group(100μL LPS and 25μg r-Stefin per mouse).All groups were administered by intraperitoneal injection.The clinical symptoms of the mice in each group were observed during the 72 hours observation period,and the survival rate curves of mice in each group were plotted at different time after LPS induction.At 12 hours and 24 hours after LPS induction,blood samples were collected from each group of mice and the contents of inflammatory cytokine in serum of mice in each group were detected by ELISA,and the liver and kidney function indexes in the serum of mice in each group were measured by fully automatic biochemistry instrument.The results showed that mice in group D and E showed dishevelled coat,reduced activity and depression after LPS induction,and mice in group F showed some improvement of the above symptoms,while mice in groups A,B and C were all healthy.The survival rate curve showed that all mice in group D died within 36 hour

关 键 词：半胱氨酸蛋白酶抑制剂 小鼠 脓毒症 脂多糖 

分 类 号：S855.9[农业科学—临床兽医学]