上海交通大学学报(医学版) ›› 2023, Vol. 43 ›› Issue (10): 1245-1254.doi: 10.3969/j.issn.1674-8115.2023.10.004

• 论著 · 基础研究 • 上一篇    

大肠埃希菌外膜囊泡对乳腺癌细胞增殖及荷瘤小鼠肿瘤生长的影响

王斓茜1,2(), 马官荣1,2, 姜咏竹1,2, 常秀林1,2, 方廖琼1, 白晋1,2()   

  1. 1.重庆医科大学生物医学工程学院,超声医学工程国家重点实验室,重庆 400016
    2.重庆医科大学重庆市生物医学工程学重点实验室,重庆 400016
  • 收稿日期:2022-12-08 接受日期:2023-06-01 出版日期:2023-08-30 发布日期:2023-08-30
  • 通讯作者: 白晋 E-mail:2962911834@qq.com;sajinbai@cqmu.edu.cn
  • 作者简介:王斓茜(1998—),女,硕士生;电子信箱:2962911834@qq.com

Effects of Escherichia coli outer membrane vesicles on proliferation of breast cancer cells and tumor growth of tumor-bearing mice

WANG Lanxi1,2(), MA Guanrong1,2, JIANG Yongzhu1,2, CHANG Xiulin1,2, FANG Liaoqiong1, BAI Jin1,2()   

  1. 1.State Key Laboratory of Ultrasound in Medicine and Engineering, College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016, China
    2.Chongqing Key Laboratory of Biomedical Engineering, Chongqing Medical University, Chongqing 400016, China
  • Received:2022-12-08 Accepted:2023-06-01 Online:2023-08-30 Published:2023-08-30
  • Contact: BAI Jin E-mail:2962911834@qq.com;sajinbai@cqmu.edu.cn

摘要:

目的·分析大肠埃希菌外膜囊泡(Escherichia coli outer membrane vesicle,E.coli-OMV)在体外对小鼠4T1乳腺癌细胞增殖的影响,在体内对BALB/c-4T1荷瘤小鼠肿瘤生长的抑制作用。方法·从E.coli的培养上清液中收集OMV并进行表征。采用荧光标记追踪法检测E.coli-OMV被4T1细胞摄入的情况。采用CCK-8法测定E.coli-OMV对4T1细胞增殖的影响。采用流式细胞术检测E.coli-OMV对4T1细胞周期的影响。利用皮下接种法建立BALB/c-4T1荷瘤小鼠模型,而后将小鼠分为E.coli-OMV组与对照(Control)组(每组10只);前者于每2 d注射0.25 mg/kg E.coli-OMV,后者则注射等剂量的PBS。观察2组小鼠的体质量、40 d生存率、肿瘤体积及质量变化,采用苏木精-伊红染色(hematoxylin-eosin staining,H-E染色)评价肿瘤组织的病理学形态,采用免疫组织化学染色法观察肿瘤组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)与细胞周期蛋白D1(CyclinD1)的表达情况。结果·E.coli-OMV是粒径为(216.00±18.30)nm的球形膜囊结构,可表达E.coli外膜蛋白A(outer membrane protein A,OmpA)与OmpC。荧光显微镜的观察结果显示4T1细胞可摄入E.coli-OMV。CCK-8法的结果显示,E.coli-OMV对4T1细胞的抑制作用与时间-剂量呈正相关。流式细胞术的结果显示,E.coli-OMV将4T1细胞的生长周期阻滞于G0/G1期。体内实验显示,与Control组相比,E.coli-OMV组小鼠的体质量在初期注射后稍有下降(P=0.031),而后出现回升;其40 d生存率增加(P=0.037);该组小鼠肿瘤的体积、质量的增长量均较少(P=0.041,P=0.004),其肿瘤体积抑制率达29.69%、肿瘤质量抑制率达49.81%。H-E染色的结果显示,E.coli-OMV组小鼠肿瘤组织细胞核分裂象较Control组减少(P=0.038)。免疫组织化学染色的结果显示,E.coli-OMV组小鼠肿瘤组织中的PCNA及CyclinD1阳性表达较Control组减少(P=0.031,P=0.002)。结论·体外研究与体内研究均表明,E.coli-OMV可对4T1细胞的增殖产生明显的抑制作用。

关键词: 细菌外膜囊泡, 乳腺癌, 细胞周期, 抑瘤作用

Abstract:

Objective ·To analyze the effect of Escherichia coli outer membrane vesicle (E.coli-OMV) on the proliferation of 4T1 breast cancer cells in vitro and the inhibition of tumor growth in BALB/c-4T1 tumor-bearing mice in vivo. Methods ·OMVs were collected from the culture supernatant of E.coli and characterized. The uptake of E.coli-OMV by 4T1 cells was detected by fluorescent label tracking method. The effect of E.coli-OMV on 4T1 cell proliferation was detected by CCK-8 method. The effect of E.coli-OMV on 4T1 cell cycle was detected by flow cytometry. The BALB/c-4T1 tumor-bearing mouse models were established by subcutaneous inoculation, and the mice were divided into E.coli-OMV group and Control group, with 10 mice in each group. The mice in the E.coli-OMV group were injected with 0.25 mg/kg E.coli-OMV every 2 d, while the mice in the Control group were injected with equal doses of PBS. The changes in body weight, 40 d survival rate, tumor volume and tumor weight of the two groups of tumor-bearing mice were observed. The pathological morphology of the tumor tissues was evaluated by hematoxylin-eosin staining (H-E staining). The expression of proliferating cell nuclear antigen (PCNA) and CyclinD1 in tumor tissues was observed by immunohistochemical staining. Results ·E.coli-OMVwas spherical membrane vesicle structure with a particle size of (216.00±18.30) nm, which expressed E.coli outer membrane protein A (OmpA) and OmpC. Fluorescence microscopy results showed that 4T1 cells could intake E.coli-OMV. CCK-8 results showed that the inhibitory effect of E.coli-OMV on 4T1 cells was positively correlated with time-dose. Flow cytometry results showed that E.coli-OMV arrested the growth cycle of 4T1 cells in G0/G1 phase. In vivo experiments showed that compared with the Control group, body weight of mice in the E.coli-OMV group decreased slightly after the initial injection (P=0.031), and then recovered, while 40 d survival rate increased (P=0.037). The growth of tumor volume and weight of mice in E.coli-OMV group were lower than those in the Control group (P=0.041, P=0.004). Its tumor volume inhibition rate reached 29.69%, and tumor weight inhibition rate reached 49.81%. The results of H-E staining showed that nuclear splitting images of tumor tissues of mice in the E.coli-OMV group decreased compared to the Control group (P=0.038). The results of immunohistochemical staining showed that the positive expression of PCNA and CyclinD1 in the tumor tissues of mice in the E.coli-OMV group decreased compared to the Control group (P=0.031, P=0.002). Conclusion ·Both in vitro and in vivo studies show that E.coli-OMV can significantly inhibit the proliferation of 4T1 cells.

Key words: bacterial outer membrane vesicle, breast cancer, cell cycle, tumor-suppressing effect

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