轮状病毒感染致腹泻乳鼠模型粪便中志贺杆菌的分离、鉴定及其对人克隆结肠腺癌细胞中紧密连接蛋白表达的影响

doi:10.13481/j.1671-587X.20210520

摘要/Abstract

摘要： 目的

探讨密封蛋白1（Claudin-1）和闭合蛋白（Occludin）在志贺杆菌感染的人克隆结肠腺癌细胞（Caco-2）中的表达情况，阐明志贺杆菌对人克隆结肠腺癌Caco-2细胞的影响及其相关机制。

方法

将10窝SPF级昆明系5日龄乳鼠分为对照组乳鼠5窝和实验组乳鼠5窝，对照组乳鼠每天每只灌胃PBS缓冲液100 μL，实验组乳鼠每天每只灌胃病毒滴度为1×10⁶PFU·mL^－1SA11株轮状病毒100 μL，采集乳鼠粪便标本通过生化实验、血清学检测及16S rRNA测序，进行志贺杆菌的分离和鉴定。将Caco-2细胞分为对照组和志贺杆菌感染组，采用不同浓度梯度的志贺杆菌感染Caco-2细胞不同时间后，台盼蓝染色检测各组细胞存活率，Real-time PCR和Western blotting法检测各组细胞中Claudin-1和Occludin mRNA及蛋白表达水平。

结果

细菌生化鉴定可观察到双糖铁培养基斜面不变色，底部为黄色，且穿刺线明显，疑似致病性肠道菌。该菌对乳糖呈阴性，对葡萄糖、麦芽糖、蔗糖和甘露醇呈阳性。16S rRNA测序，与志贺杆菌序列同源性为99%，确定为志贺杆菌。与对照组比较，志贺杆菌感染组志贺杆菌感染4 h内细胞存活率差异无统计学意义（P>0.05）：与1×10⁶CFU·mL^－1志贺杆菌共培养3 h时，Caco-2细胞中Claudin-1和Occludin mRNA及蛋白表达水平明显降低（P<0.05）。

结论

通过轮状病毒感染致腹泻乳鼠模型粪便中分离的志贺杆菌可降低人克隆Caco-2中Occludin和Claudin-l的表达水平，提高肠上皮细胞的通透性，从而影响肠上皮细胞的正常屏障功能。

关键词: 志贺杆菌, Caco-2细胞, 密封蛋白1, 闭合蛋白

Abstract: Objective

To investigate the expressions of tight junction proteins Claudin-1 and Occludinin in the human colon adenocarcinoma cells （Caco-2） infected by Shigella， and to elucidate the effect of Shigella on the human colon adenocarcinoma Caco-2 cells and its related mechanism.

Methods

The ten nests of SPF Kunming 5-day-old suckling mice were divided into control group and experimental group，with 5 nests in each group. Each suckling mouse in control group was given 100 μL PBS per day，the mouse in experimental group was given 100 μL 1×10⁶ PFU·mL^－1 SA11 strain rotavirus.The fecal samples of suckling mice were collected，and Shigella was isolated and identified by biochemical test， serological detection and 16S rRNA sequencing.The Caco-2 cells were divided into control group and Shigella infection group. Different concentrations of Shigella were used to infect the Caco-2 cells for different time. The cell survival rates in various groups were detected by trypan blue staining， and the expression levels of Claudin-1 and Occludin mRNA and proteins in the cells in various groups were detected by Real-time PCR and Western blotting methods.

Results

The biochemical identification of bacteria showed that the color of inclined surface of disaccharide iron medium did not change， the bottom was yellow， and the puncture line was obvious， which was suspected of pathogenic intestinal bacteria. It was negative for lactose and positive for glucose， maltose， sucrose and mannitol.The 16S rRNA sequencing results showed 99% homology with Shigella， and it was identified as Shigella. Compared with control group， there was no significant difference in the cell survival rate within 4 h of infection in Shigella group （P>0.05）； when cocultured with 1×10⁶ CFU·mL^－1Shigella for 3 h， the expression levels of Claudin-1 and Occludin mRNA and proteins in the Caco-2 cells were decreased significantly （P<0.05）.

Conclusion

Shigella isolated from feces of diarrhea model mice infected with rotavirus can reduce the expression levels of Occludin and Claudin-1 in the human colon adenocarcinoma cells（Caco-2）， improve the permeability of intestinal epithelial cells， and affect the normal barrier function of intestinal epithelial cells.

Key words: Shigella, Caco-2 cells, Claudin-1, Occludin

中图分类号:

R378.25

于美玲,陆恒章,陶晓莉,程美慧,李永刚,赵微. 轮状病毒感染致腹泻乳鼠模型粪便中志贺杆菌的分离、鉴定及其对人克隆结肠腺癌细胞中紧密连接蛋白表达的影响[J]. 吉林大学学报(医学版), 2021, 47(5): 1221-1228.

Meiling YU,Hengzhang LU,Xiaoli TAO,Meihui CHENG,Yonggang LI,Wei ZHAO. Isolation and identification of Shigella from feces of rotavirus infected diarrhea suckling mice and its effect on tight junction protein expression in human colon adenocarcinoma cells[J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1221-1228.

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Group	Survival rate
Group	(t/h) 3	4	5	6
Control	80.00±2.50	84.00±4.50	83.00±2.45	83.00±2.45
Shigella(CFU·mL^－1)
1×10⁵	84.00±5.35	84.00±5.44	77.00±2.83	71.00±4.90
1×10⁶	78.00±2.06	91.00±0.82	62.00±2.50^*	70.00±0.47
1×10⁷	75.00±2.63	87.00±6.60	67.00±3.09	68.00±2.36^*
1×10⁸	72.00±1.63	86.00±4.97	50.00±6.38^*	57.00±2.50^*

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