粪肠球菌在体外细胞模型和乳鼠轮状病毒感染模型中对轮状病毒SA11株复制的抑制作用

doi:10.13481/j.1671-587X.20230117

摘要/Abstract

摘要：

目的探讨加入粪肠球菌后轮状病毒SA11株在体内和体外的复制情况，为阐明粪肠球菌影响轮状病毒感染提供依据。方法体外实验，将10⁸~10¹² CFU·mL^－1粪肠球菌进行10倍浓度梯度稀释，与Caco-2细胞共培养12、18、24、30和48 h，同时设对照组（Caco-2细胞感染轮状病毒后不加入粪肠球菌），采用CCK-8法检测不同浓度粪肠球菌作用后各组Caco-2细胞存活率；感染复数（MIO）值为0.1的轮状病毒作用于Caco-2细胞1 h后加入10⁸~10¹² CFU·mL^－1粪肠球菌培养18 h，同时设对照组，实时荧光定量PCR（RT-qPCR）法检测各组Caco-2细胞中轮状病毒VP6基因拷贝数，免疫荧光灶法检测各组Caco-2细胞中轮状病毒滴度。体内实验，采用18窝4~5日龄SPF级昆明系乳鼠，相同窝数随机分为对照组、抗生素处理组和粪肠球菌移植组，抗生素处理组和粪肠球菌移植组乳鼠先用四联抗生素灌胃3 d耗竭其肠道菌群，然后3组乳鼠开始每日灌胃10⁷ FFU·mL^－1轮状病毒100 μL，持续8 d，粪肠球菌移植组乳鼠在灌胃病毒同时灌胃10¹⁰CFU·mL^－1粪肠球菌100 μL，收取病毒感染第2、4、6和8天各组乳鼠粪便，RT-qPCR法检测各组乳鼠粪便中轮状病毒拷贝数。结果体外实验， CCK-8法检测，与对照组比较，不同浓度粪肠球菌作用48 h内各组Caco-2细胞存活率均有不同程度升高，表明粪肠球菌在该浓度范围内对Caco-2细胞无毒性；RT-qPCR法检测，与对照组比较，10¹⁰、10¹¹和10¹²CFU·mL^－1粪肠球菌组Caco-2细胞中轮状病毒VP6基因拷贝数分别降低了75.8%、99.9%和99.9%（P<0.05）；免疫荧光灶法检测，与对照组比较，10⁸、10¹⁰和10¹²CFU·mL^－1粪肠球菌组Caco-2细胞中轮状病毒滴度分别降低13%、77%和100%（P<0.05）。体内实验，与对照组比较，粪肠球菌移植组乳鼠粪便中病毒基因拷贝数明显减少（P<0.05）。结论在体外细胞模型和乳鼠感染轮状病毒模型中粪肠球菌对轮状病毒的复制均发挥抑制作用。

关键词: 粪肠球菌, 轮状病毒, 菌群移植, 益生菌

Abstract:

Objective To explore the replication of rotavirus SA11 strain in vivo and in vitro after adding Enterococcus faecalis （E. faecalis），and to provide the evidence for clarifying the effect of E. faecalis on rotavirus infection. Methods In in vitro experiment， 10⁸－10¹² CFU ·mL^－1E. faecalis were diluted with 10 times concentration gradient and co-cultured with Caco-2 cells for 12， 18， 24， 30，and 48 h，and control group （Caco-2 cells infacted with rotavius without the addttion of E. faecalis） was set up at the same time. The survival rates ofCaco-2 cellsin various groups after treatment of different concentrations of E. faecalis were determined by CCK-8 method.One hour after the Caco-2 cells were treated with rotavirus with multiplicity of infection （MIO） value of 0.1，they were cultured with 10⁸－10¹² CFU·mL^－1E.faecalis for 18 h. The rotavirus VP6 gene copy numbers of Caco-2 cells in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method and the rotavirus titers of Caco-2 cells in various groups were detected by immunofluorescence focus method.In in vivo experniment， 18 litters of 4－5 d old SPF Kunming suckling mice were randomly divided into control group， antibiotic treatment group and E. faecalis transplantation group. The suckling mice in antibiotic treatment group and E. faecalis transplantation group were gavaged with quadruple antibiotics for 3 d to deplete their intestinal flora， and the suckling mice in three groups received 10⁷FFU·mL^－1 rotavirus 100 μL by gavage daily for 8 d， and the suckling mice in E. faecalis transplantation group were given 10¹⁰ CFU·mL^－1E. faecalis 100 μL at the same time.The feces were collected on the 2nd，4th，6th，and 8th days after virus infection， and the copy numbers of rotavirus in feces were measured by RT-qPCR method. Results In in vitro experiment，the CCK-8 method results showed that compared with control group，the survival rates of Caco-2 cells in various groups in 48 h of treatment of different concentrations of E. faecalis were increased（P<0.05）， indicating that E. faecalis was non-toxic to the Caco-2 cells within this concentration range.The RT-qPCR results showed that compared with control group，the number of rotavirus gene copies in the Caco-2 cells in 10¹⁰， 10¹¹，and 10¹²CFU·mL^－1E. faecalis groups weredecreased by 75.8%， 99.9%， and 99.9%，respectively（P<0.05）. The results of immunofluorescence focus detection showed that compared with control group，the rotavirus titer in 10⁸，10¹⁰，and 10¹²CFU·mL^－1E. faecalis groups were decreased by 13%，77%， and 100%， respectively （P<0.05）.In in vivo experiment， compared with control group， the copy number of fecal virus gene of the suckling mice in E. faecalis transplantation group was significantly decreased（P<0.05）. Conclusion In in vitro cell model and rotavirus infection model of suckling mice， E. faecalis can inhibit the replication of rotavirus.

Key words: Enterococcus faecalis, Rotavirus, Flora transplantation, Probiotics

中图分类号:

R373.2

刘洋,于美玲,程美慧,刘长城,贾雪娇,刘梦琦,李永刚,赵微. 粪肠球菌在体外细胞模型和乳鼠轮状病毒感染模型中对轮状病毒SA11株复制的抑制作用[J]. 吉林大学学报(医学版), 2023, 49(1): 131-138.

Yang LIU,Meiling YU,Meihui CHENG,Changcheng LIU,Xuejiao JIA,Mengqi LIU,Yonggang LI,Wei ZHAO. Inhibitory effect of Enterococcus faecalis on rotavirus SA11 strain replication in in vitro cell model and suckling rat rotavirus infection model[J]. Journal of Jilin University(Medicine Edition), 2023, 49(1): 131-138.

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Group	Survival rate
Group	(t/h) 12	18	24		30	48
Control	100±0	100±0	100±0	100±0		100±0
E. faecalis (CFU·mL^－1)
10⁸	124±9	113±6	88±2^*	109±3^*		113±5^*
10⁹	122±9	114±3^*	131±2^*	105±2^*		104±1
10¹⁰	134±10^*	109±5	101±2	109±3^*		132±5^*
10¹¹	127±9	113±4^*	127±9	119±9^*		131±9^*
10¹²	127±9	115±5^*	121±1^*	110±1^*		99±9

Group	Copy number
Control E. faecalis(CFU·mL^－1)	2.72×10¹⁰±794 874 336.7
10⁸	3.39×10¹⁰±2 081 066 348.0
10⁹	3.13×10¹⁰±1 150 565 810.0
10¹⁰	6.57×10⁹±522 776 021.0^*
10¹¹	7.08×10⁶±1 356 982.9^*
10¹²	1.23×10⁷±1 177 083.8^*

Group	Immunofluorescencenumber（×10）	Virus titer（FFU·mL^－1）
Contorl	126±15	2.510×10⁶±315 246
Negative contorl E. faecalis (CFU·mL^－1)	0	0
10⁸	109±8^*	2.178×10⁶±126 233^*
10¹⁰	29±3^**	5.780×10⁵±63 248^**
10¹²	0	0

Group	Copy number
Group	(t/d) 2	4	6	8
Control	1.12×10¹⁰±212 892 900	3.36×10⁹±45 211 863	4.38×10⁸±11 511 474	8.65×10⁷±3 762 326
Antibiotic treatment	3.07×10⁹±234 541 616^**	9.93×10⁸±552 293 962	2.49×10⁸±32 036 586^*	1.32×10⁷±2 372 030^**
E. faecalis transplantation	2.43×10⁸±11 306 407^**	2.75×10⁸±23 749 087^**	8.49×10⁷±4 189 221^**	1.62×10⁷±3 045 903^**
^P<0.05，^*P<0.01 compared with control group.

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