In vitro experimental study on lymphangiogenesis mediated by colorectal cancer-associated regulatory B cells
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摘要:
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，BregCT26诱导组［（4.90±0.05）% vs. （27.63±1.12）%］和BregMC38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。 Abstract: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 BregCT26-induced groups [(4.90±0.05) vs. (27.63±1.12)] and BregMC38-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. -
Key words:
- regulatory B cell (Breg) /
- colorectal cancer /
- lymphangiogenesis /
- lymphatic metastasis
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图 3 流式细胞术检测结肠癌相关性Breg细胞比例
A：各组Breg细胞的流式图；B：24 h共培养体系Breg比例的统计图；C：48 h共培养体系Breg比例的统计图；**：P<0.01（与空白对照组相比），ns：差异无统计学意义（与阳性对照组LPS+Anti-CD40相比）
图 4 ELISA检测结肠癌相关性Breg细胞条件培养基的细胞因子浓度
A：共培养体系中各组VEGF-A分泌水平统计图；B：共培养体系中各组VEGF-C分泌水平统计图；C：共培养体系中各组VEGF-D分泌水平统计图；*：P<0.05，**：P<0.01
图 5 Breg对淋巴结内皮细胞管腔形成能力的影响
A：荧光显微镜下观察各组SVEC4-10在基质胶上形成的管腔样结构（×200）；B：Image J分析相对管腔长度；C：Image J分析相对管腔节点数；ns：差异无统计学意义，**：P<0.01（与空白组相比）；##：P<0.01（与B细胞组相比）
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