低浓度猪链球菌溶血素诱导猪肾细胞凋亡方式研究  

Study of Apoptosis Mode of Porcine Kidney Cells Induced by Low Concentration Suilysin

作　　者：戴世馨 赵勤 徐魁 牟欣然 杜森焱 伍锐 文翼平 黄小波 颜其贵 曹三杰 DAI Shi-Xin;ZHAO Qin;XU Kui;MOU Xin-Ran;DU Sen-Yan;WU Rui;WEN Yi-Ping;HUANG Xiao-Bo;YAN Qi-Gui;CAO San-Jie(College of Veterinary Medicine/Research Center of Swine Diseases/National Demonstration Center for Experimental Animal Education/Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique,Ministry of Agriculture and Rural Affairs,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院/猪病研究中心/国家级动物类实验教学示范中心/农业农村部兽用药物与兽医诊断技术四川科学观测站,成都611130

出　　处：《农业生物技术学报》2024年第2期355-366,共12页Journal of Agricultural Biotechnology

基　　金：四川省“十四五”川猪重大科技专项(2021ZDZX0010);中央引导地方科技发展专项(2021ZYD0086)。

摘　　要：猪链球菌(Streptococcus suis)是一种危害严重的人畜共患病原菌,猪链球菌溶血素(suilysin,Sly)在猪链球菌的侵袭和致病中起关键作用,对多种类型的细胞具有毒性作用,但目前关于Sly的细胞毒性机制研究较少。为了探讨低浓度(0.3~0.6μg/mL)Sly诱导猪肾细胞PK-15(porcine kidney-15)的死亡方式,本研究首先采用原核表达并纯化获得重组猪链球菌溶血素(recombinant suilysin,rSly),通过乳酸脱氢酶(lactate dehydrogenase,LDH)释放检测rSly对PK-15细胞的毒性作用,并针对4种常见程序性细胞死亡方式,利用qPCR、间接免疫荧光、Werstern blot和流式细胞术对rSly作用于PK-15细胞12及24 h诱导的细胞死亡方式进行验证。结果显示,成功表达具有较高溶血活性(溶血单位为214)的rSly毒素蛋白,确定其对PK-15细胞的毒性作用呈浓度依赖性,并且发现rSly作用PK-15细胞12和24 h均引起PK-15细胞凋亡相关基因—半胱氨酸天冬氨酸蛋白酶3(cysteine aspartic acid specific protease 3,Caspase 3)、Caspase 9的转录水平显著升高(P<0.05),细胞凋亡率与r Sly浓度呈正相关,0.6μg/mL rSly作用PK-15细胞24 h,细胞凋亡率从9.35%增至49.9%;自噬关键基因—微管相关蛋白轻链3B(microtubule associated protein 1 light chain 3β,LC3β)基因转录水平无明显变化且无自噬小体形成;焦亡关键基因—胃泌素D(gasdermin D,GSDMD)的转录水平无明显变化且GSDMD蛋白未发生明显切割;坏死性凋亡相关基因下调,且磷酸化混合系列蛋白激酶样结构域蛋白(phosphorylated mixed lineage kinase domain-like protein,p-MLKL)表达水平受到抑制。上述结果提示低浓度rSly诱导PK-15细胞死亡的方式主要是凋亡。本研究丰富了Sly毒性作用的细胞模型,有助于揭示参加猪链球菌感染发展的分子机制。Streptococcus suis is a serious zoonotic pathogen.Suilysin(Sly)plays a key role in the invasion and pathogenesis of S.suis.It has toxic effects on various types of cells,but there are few studies on the cytotoxic mechanism of Sly.In order to explore the death modes of the porcine kidney cells 15(PK-15)induced by low concentration of Sly(0.3~0.6μg/mL),in this study,recombinant suilysin(rSly)was obtained by prokaryotic expression and purification,the toxicity of rSly on PK-15 cells was detected by lactate dehydrogenase(LDH)release assay.Then,for 4 common programmed cell death modes,qPCR,immunofluorescence,Western blot,and flow cytometry assay were used to verify the ways of rSly-induced cell death in PK-15 cells for 12 and 24 h.The results showed that rSly with high hemolytic activity(214 hemolytic units)was successfully expressed,and its toxic effect on PK-15 cells was confirmed to be concentration-dependent.The transcription levels of apoptosis-related genes-cysteine aspartic acid specific protease 3(Caspase 3)and Caspase 9 were significantly increased after rSly treatment for 12 and 24 h(P<0.05).The apoptosis rate was positively correlated with the concentration of rSly,and the apoptosis rate of PK-15 cells increased from 9.35% to 49.9% after 0.6μg/mL rSly treatment for 24 h.There was no significant change in the transcription level of microtubule associated protein 1 light chain 3β(LC3β),a key autophagy gene,and no autophagosome formation.The transcription level of gasdermin D(GSDMD),a pyroptosis-related gene had no significant change,and no obviously cleavage of GSDMD protein.Necroptosis-related genes were down-regulated,and the expression level of phosphorylated mixed lineage kinase domain-like protein(p-MLKL)was inhibited.The results suggested that low concentration of rSly induced PK-15 cell death mainly by apoptosis.This study enriches the cellular model of Sly toxicity and helps to reveal the molecular mechanisms underlying the development of S.suis infection.

关 键 词：重组猪链球菌溶血素(rSly) 猪肾细胞(PK-15) 细胞毒性 细胞死亡方式 细胞凋亡 

分 类 号：S855.1[农业科学—临床兽医学]