人α1抗胰蛋白酶的分离纯化及其对C-BSA型大鼠肾炎模型的影响  

Isolation and purification of alpha 1-antitrypsin and its effect on C-BSA rat nephritis model

作　　者：孙兆健 蒋伟[1] 张英[1] 田建明[2] 王妍[1] SUN Zhao-jian;JIANG Wei;ZHANG Ying;TIAN Jian-ming;WANG Yan(College of Applied Chemistry and Biotechnology,Shenzhen Polytechnic,Shenzhen 518055,Guangdong Province,China;不详)

机构地区：[1]深圳职业技术学院应用化学与生物技术学院,广东深圳518055 [2]吉林省中医药科学院,吉林长春130021

出　　处：《中国生物制品学杂志》2020年第11期1247-1252,共6页Chinese Journal of Biologicals

基　　金：深圳市科技创新委项目(JCYJ20160527172247868)。

摘　　要：目的分离纯化α1抗胰蛋白酶(alpha 1-antitrypsin,AAT),并评价其对C-BSA型大鼠肾炎模型的影响。方法以Cohn组分Ⅳ(Cohn frcationⅣ,FⅣ)为原料,采用Q Sepharose Fast Flow阴离子交换层析和S-200凝胶过滤柱层析纯化AAT,底物显色法检测其生物学活性。建立阳离子化小牛血清蛋白型大鼠肾炎模型,分别注射5.75和11.5 mg/kg的AAT,同时设正常组(未建模),隔日经大鼠腹腔注射给药1次,连续注射4周,模型组和正常组注射等体积的PBS。检测各组大鼠的尿常规及24 h尿液总蛋白量,并对肾脏组织进行病理学检查,同时检测肾脏中IgG沉积及AAT、Desmin、Nephrin、Podocin的表达情况。结果纯化的AAT纯度为96%,浓度为11.85 mg/mL,活性回收率为47.67%,比活为5.37。与模型组比较,高剂量AAT组大鼠24 h尿蛋白量明显降低(P<0.05),尿常规部分指标有所改善,肾小球体积增大程度有所减轻;高、低剂量AAT组大鼠肾小球IgG沉积均略减少(P>0.05),肾小球与肾小管AAT阳性表达量明显增加(P<0.001),肾小球Desmin蛋白有减轻趋势,但差异无统计学意义(P>0.05);高剂量AAT组大鼠肾小球Nephrin及Podocin蛋白均明显增多(P<0.05),低剂量AAT组略有升高趋势,但差异无统计学意义(P>0.05)。结论本纯化工艺可获得纯度较高的AAT,其对大鼠肾炎具有一定提高疗效的作用。Objective To isolate and purify the alpha 1-antitrypsin(AAT)and evaluate its effect on cationic bovine serum protein(C-BSA)rat nephritis model.Methods AAT was purified from Cohn fraction Ⅳ by Q Sepharose Fast Flow anion exchange chromatography and S-200 size exclusion chromatography,and determined for biological activity by substrate color development method.C-BSA rat nephritis model was established,and the model rats were injected i.p.with AAT at dosages of 5.75 and 11.5 mg/kg respectively every other day for 4 weeks,while those in model control and normal control groups with PBS at the same volumes.The rats in various groups were subjected to routine urine test and determination of total urine protein content in 24 h,of which the kidneys were subjected to pathological examination,while the IgG sediment as well as expressions of AAT,Desmin Nephrin and Podocin in kidney were determined.Results The purity,concentration,activity recovery and specific activity of purified AAT were 96%,11.85 mg/mL,47.67% and 5.37 respectively.Compared with those in model control group,the total urine protein content in 24 h of rats injected with AAT at high dosage decreased significantly(P<0.05),while partial indicators in routine urine test were improved,the glomerular enlargement was relieved.The IgG sediment in glomerulus of rats injected with AAT high and low dosages decreased slightly(P>0.05),while the positive expressions of AAT in glomerulus and tubules increased significantly(P<0.001),and that of Desmin protein in glumerulus decreased insignificantly(P>0.05).Both Nephrin and Podocin proteins in glumerulus of rats injected with AAT at a high dosage increased significantly(P<0.05),while those at a low dosage increased insignificantly(P>0.05).Conclusion Highly purified AAT was obtained by the procedure,which enhanced the curative effect on nephritis in rats at a certain degree.

关 键 词：Α1抗胰蛋白酶 Cohn组分Ⅳ 分离纯化 大鼠 肾炎 

分 类 号：R-332[医药卫生] R692.3