结肠癌相关性调节性B细胞介导淋巴管生成的体外研究

In vitro experimental study on lymphangiogenesis mediated by colorectal cancer-associated regulatory B cells

目的:通过共培养模型,验证结肠癌相关性Breg细胞促进淋巴管生成的作用及相关机制。方法:利用结肠癌细胞CT26及MC38与B细胞构建共培养模型,诱导结肠癌相关性Breg细胞,流式细胞术鉴定CD19+IL-10+Breg细胞比例。ELISA法检测结肠癌相关性Breg细胞条件培养基中VEGF-A、VEGF-C和VEGF-D的分泌水平。利用划痕实验及管腔形成实验检测Breg条件培养基干预后淋巴结内皮细胞SVEC4-10的迁移及管腔形成能力。结果:与24 h共培养体系相比,Breg^(CT26)诱导组[(4.90±0.05)%vs.(27.63±1.12)%]和Breg^(MC38)诱导组[(5.85±0.86)%vs.(24.27±2.27)%]在48 h共培养体系中能诱导更多的Breg细胞,且结肠癌相关性Breg诱导组VEGF-A和VEGF-C的表达水平与空白组相比显著增加(P<0.05)。管腔形成实验及划痕实验结果表明,结肠癌相关性Breg细胞能增强SVEC4-10的成管和迁移能力。结论:在肿瘤微环境中,结肠癌细胞通过诱导B细胞向Breg细胞转化,上调VEGF-A和VEGF-C分泌水平,增强SVEC4-10的成管和迁移能力,从而促进肿瘤淋巴管生成。

Objective:To investigate the role and mechanism of action of colorectal cancer-associated regulatory B cells(Breg)in promoting lymphangiogenesis using a coculture model.Methods:The colorectal cancer cells,CT26 and MC38,were cocultured with B cells to induce colorectal cancer-associated Breg cells;the proportion of CD19+IL-10+Breg cells was determined using flow cytometry;the secretion levels of VEGF-A,VEGF-C,and VEGF-D in conditioned medium of colorectal cancer-associated Breg cells were detected using ELISA;scratch and tube formation assays were used to detect the migration and tube formation abilities of the lymph node endothelial cell line,SVEC4-10,after intervention of Breg-conditioned medium.Results:Breg^(CT26)-induced groups[(4.90±0.05)vs.(27.63±1.12)]and Breg^(MC38)-induced groups[(5.85±0.86)vs.(24.27±2.27)]from the 48h coculture system induced more Breg cells than those from the 24h coculture system;the expression levels of VEGF-A and VEGF-C in colorectal cancer-associated Breg cells were significantly higher than those in cells from the blank group(P<0.05).Moreover,colorectal cancer-associated Breg cells enhanced both the tube formation and migration abilities of SVEC4-10 cells.Conclusions:In the tumor microenvironment,colorectal cancer cells induce B cell transformation into Breg cells.This change upregulates the secretion levels of VEGF-A and VEGF-C,enhances the tube formation and migration abilities of SVEC4-10,and promotes tumor lymphangiogenesis.