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

doi:10.3969/j.issn.1674-8115.2023.10.004

摘要/Abstract

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

中图分类号:

R737.9

王斓茜, 马官荣, 姜咏竹, 常秀林, 方廖琼, 白晋. 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[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1245-1254.

WANG Lanxi, MA Guanrong, JIANG Yongzhu, CHANG Xiulin, FANG Liaoqiong, BAI Jin. Effects of Escherichia coli outer membrane vesicles on proliferation of breast cancer cells and tumor growth of tumor-bearing mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(10): 1245-1254.

图/表 10

图1 E.coli-OMV的一般特征分析Note： A. Transmission electron micrograph of E.coli-OMV. B. Particle size distribution of E.coli-OMV. C. Detection of outer membrane marker proteins in E.coli-OMV by Western blotting.

Fig 1 Analysis of the characterization of E.coli-OMV

图2 4T1细胞对 E.coli-OMV的摄入观察（×100）Note： Green fluorescence indicates PKH67-labelled E.coli-OMV, and white arrows indicate E.coli-OMV in the nucleus.

Fig 2 Observation on the uptake of E.coli-OMV by 4T1 cells (×100)

图3 CCK-8法检测 E.coli-OMV对4T1细胞增殖的影响Note： A. Effect of the different concentrations of E.coli-OMV on D(450 nm) of 4T1 cells at different time points. B. Analysis of the inhibition rate of E.coli-OMV at different concentrations on 4T1 cell proliferation under different time points.

Fig 3 Effect of E. coli-OMV on 4T1 cells proliferation by CCK-8 method

图4 流式细胞术检测 E.coli-OMV对4T1细胞周期分布的影响Note： A. Flow assay graphs of 4T1 cell cycle. B. Effect of different concentrations of E.coli-OMV on 4T1 cell cycle distribution at different time points.

Fig 4 Effect of E. coli-OMV on 4T1 cell cycle distribution by flow cytometry

图5 2组BALB/c-4T1荷瘤小鼠的体质量变化

Fig 5 Changes in body weight of BALB/c-4T1 tumor-bearing mice in the two groups

图6 2组BALB/c-4T1荷瘤小鼠的生存率分析

Fig 6 Survival rate analysis of BALB/c-4T1 tumor-bearing mice in the two groups

图7 2组BALB/c-4T1荷瘤小鼠的肿瘤体积变化Note： A. Tumor volume growth curve of the mice injected from 0 d to 20 d. B. Tumor volume growth in mice at d 20 post-injection.

Fig 7 Changes in tumor volume growth of BALB/c-4T1 tumor-bearing mice in the two groups

图8 2组BALB/c-4T1荷瘤小鼠的肿瘤质量变化Note： A. Anatomy picture of tumor tissues in the two groups at 20 d post-injection. B. Statistical analysis of tumor weight in the two groups at 20 d post-injection.

Fig 8 Changes in tumor weight of BALB/c-4T1 tumor-bearing mice in the two groups

图9 2组BALB/c-4T1荷瘤小鼠的肿瘤组织的H-E染色分析（×200）Note： A. H-E staining of tumor tissues. White arrows indicate pathological nuclear schizophrenia. B. Statistical analysis of pathological nuclear schizophrenia of tumor tissues. HPF—High power field.

Fig 9 Analysis of H-E staining of tumor tissues of BALB/c-4T1 tumor-bearing mice in the two groups (×200)

图10 免疫组织化学染色分析2组BALB/c-4T1荷瘤小鼠肿瘤组织中PCNA、CyclinD1的表达（×200）Note： A. Immunohistochemical images of PCNA and CyclinD1 expressions in the two groups. The areas circled by the red dotted line a、b、c are large area of positive expression. Red arrows indicate scattered nuclei with positive expression. B. Statistical analysis of mean optical density of PCNA and CyclinD1 positive staining.

Fig 10 Analysis of the expression of PCNA and CyclinD1 in tumor tissues of BALB/c-4T1 tumor-bearing mice in the two groups by immunohistochemical staining (×200)

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