罗伊氏乳杆菌对轮状病毒体内外复制的抑制作用及其对免疫因子表达的影响  

Inhibitory effect of Lactobacillus reuteri on rotavirus replication in vivo and in vitro and its effect on expression of immune factors

作　　者：李小凤 程美慧 刘洋 刘长城 贾雪娇 刘梦琦 赵微 LI Xiaofeng;Cheng Meihui;LIU Yang;LIU Changcheng;JIA Xuejiao;LIU Mengqi;ZHAO Wei(Laboratory of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China;Department of Laboratory,Central Hospital,Huangshi City,Hubei Province,Huangshi 435000,China)

机构地区：[1]锦州医科大学基础医学院病原生物学实验室,辽宁锦州121000 [2]湖北省黄石市中心医院检验科,湖北黄石435000

出　　处：《吉林大学学报（医学版）》2024年第6期1597-1605,共9页Journal of Jilin University：Medicine Edition

基　　金：辽宁省教育厅基金项目(LJKMZ20221243);辽宁省科技厅科技计划联合计划基金项目(2023-MSLH-046)。

摘　　要：目的:探讨罗伊氏乳杆菌对轮状病毒(RV)SA11株体内外复制的抑制作用,并阐明其对相关免疫因子表达的影响。方法:体外实验,培养并鉴定罗伊氏乳杆菌,绘制罗伊氏乳杆菌标准曲线和生长曲线,筛选罗伊氏乳杆菌培养最佳时间和最适浓度。采用5×10^(8)、10×10^(8)、50×10^(8)、100×10^(8)、200×10^(8)和500×10^(8)CFU·mL^(-1)罗伊氏乳杆菌感染细胞,台盼蓝染色法检测Caco-2细胞存活率。将不同浓度罗伊氏乳杆菌与RV体外共孵育并作用于Caco-2细胞,Caco-2细胞分为阴性对照组(NC组)、阳性对照组(PC组)和10~7、10^(8)、10~9及1010CFU·mL^(-1)罗伊氏乳杆菌组,免疫荧光灶法检测罗伊氏乳杆菌作用后Caco-2细胞中病毒滴度,实时荧光定量PCR(RT-qPCR)法检测不同浓度罗伊氏乳杆菌作用后Caco-2细胞中RV VP6基因拷贝数。体内实验,将25窝SPF级乳鼠分为对照组、RV组(感染SA11毒株)、Ab-NC组(抗生素处理耗竭肠道菌群)、Ab-RV组(耗竭肠道菌群后感染SA11毒株)和Ab-Lac-RV组(耗竭肠道菌群,并感染罗伊氏乳杆菌后感染SA11毒株)。收取各组乳鼠灌胃第2、4、6、8和10天的粪便样本和灌胃第4天结肠组织样本,RT-qPCR法检测各组乳鼠粪便中RV VP6基因拷贝数和结肠组织中白细胞介素(IL)-1β、IL-8、IL-10、γ干扰素(IFN-γ)和肿瘤坏死因子(TNF)-αmRNA表达水平。结果:罗伊氏乳杆菌生长良好,形态圆润,呈圆形、光滑和乳白色的凸起样菌落,且边缘较整齐;革兰染色后菌体呈紫色、不规则和方形杆状;经16SrDNA测序后序列同源性为99%,提示罗伊氏乳杆菌活化成功;罗伊氏乳杆菌活菌数与吸光度(A)值呈线性关系,回归分析标准曲线为Y=0.4375X+0.0006,R2=0.9994。培养0~2 h,细菌处于对数生长期,细菌生长迟缓;培养2~14 h,细菌快速生长,并于培养14~16 h时细菌生长趋于稳定,培养16 h时达到细菌生长速率顶峰,随后进入衰亡期。5×10^(8)、10×10^(8)、50×10^(8)、100×10^(8)�Objectives:To discuss the inhibitory effect of Lactobacillus reuteri on the replication of rotavirus(RV)strain SA11 in vivo and in vitro,and to clarify its effect on the expression of related immune factors.Methods:For in vitro experiments,Lactobacillus reuteri was cultured and identified,and the standard curve and growth curve were plotted to screen the optimal time and concentration for Lactobacillus reuteri cultivation.The cells were infected with Lactobacillus reuteri at the concentrations of 5×10^(8),10×10^(8),50×10^(8),100×10^(8),200×10^(8),and 500×10^(8)CFU·mL−1,and the surival rates of Caco-2 cells were detected by trypan blue staining method.Various concentrations of Lactobacillus reuteri were co-incubated with RV in vitro and applied to the Caco-2 cells.The cells were divided into negative control group(NC group),positive control group(PC group),and 107,10^(8),109,and 1010 CFU·mL−1 Lactobacillus reuteri groups.Immunofluorescence focus method was used to detect the viral titers in the Caco-2 cells after treated with Lactobacillus reuteri and real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the copy numbers of RV VP6 gene in the Caco-2 cells after treated with various concentrations of Lactobacillus reuteri.In in vivo experiments,25 litters of SPF suckling mice were divided into control group,RV group(infected with SA11 strain),Ab-NC group(treated with antibiotic to deplete gut microbiota),Ab-RV group(depleting gut microbiota and then infected with SA11 strain),and Ab-Lac-RV group(depleting gut microbiota,treated with Lactobacillus reuteri,and then infected with SA11 strain).The fecal samples were collected on days 2,4,6,8,and 10 gavage,colon tissue sample were collected on day 4 of and RT-qPCR method was used to detect the copy numbers of RV VP6 gene in feces and the mRNA expression levels of interleukin(IL)-1β,IL-8,IL-10,interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α)in colon tissue of the suckling mice in vartious groups.Results:The Lactobacillus reuteri

关 键 词：罗伊氏乳杆菌 轮状病毒 乳鼠 免疫因子 标准曲线 

分 类 号：R373.2[医药卫生—病原生物学]