胆囊癌相关性成纤维细胞促进裸鼠足垫肿瘤淋巴结转移的体内研究

In vivo study of gallbladder cancer-associated fibroblast promoting tumor lymph node metastasis in foot pad of nude mice

目的构建体内裸鼠足垫肿瘤转移模型,探讨肿瘤相关性成纤维细胞(cancer-associated fibroblast,CAF)对胆囊癌淋巴转移的影响。方法收集病人胆囊癌和正常胆囊组织标本,通过酶消化法提取原代CAF和正常成纤维细胞(normal fibroblast,NF)。利用免疫荧光实验和蛋白质印迹法(Western blot)鉴定CAF和NF;构建裸鼠足垫肿瘤转移模型,随机分为两组:CAF+NOZ组和NF+NOZ组,将相应的细胞植入小鼠足垫,通过活体动物成像系统定期观察记录同侧腘窝淋巴结转移情况。4周后,获取淋巴结并进行苏木精-伊红(hematoxylin and eosin,HE)染色和免疫组化分析肿瘤淋巴结转移情况。结果从病人组织中成功提取出CAF和NF。免疫荧光结果显示α平滑肌肌动蛋白(α-SMA)在CAF中显著高表达,并且Western blot结果提示CAF中标志蛋白α-SMA和FAP明显高表达。在裸鼠足垫肿瘤模型中发现,相较于NF组,CAF组腘窝淋巴结明显肿大,差异具有统计学意义(P<0.05)。HF染色和细胞角蛋白-7(cytokeratin 7,CK7)的免疫组化染色发现,CAF组阳性淋巴结比率明显增加,差异具有统计学意义(P<0.05),并且CK7表达明显增高。结论通过构建裸鼠足垫肿瘤转移模型,证实CAF可以促进胆囊癌的淋巴转移。

Objective To investigate the effect of cancer-associated fibroblast(CAF)on lymph node metastasis of gallbladder cancer by using a model of lymphatic metastasis of foot pad tumor in nude mice.Methods Tissue samples of human gallbladder cancer and normal gallbladder were collected.Primary CAF and normal fibroblast(NF)were isolated by enzymatic digestion method.CAF and NF were identified by immunofluorescence assay and Western blot.The tumor metastasis model of foot pad in nude mice was constructed and randomly divided into two groups:CAF+NOZ group and NF+NOZ group,and the corresponding cells were implanted into the foot pad of mice,and the metastasis of ipsilateral popliteal lymph node was regularly observed and recorded by the live animal imaging system.After 4 weeks,lymph nodes were obtained and analyzed for tumor lymph node metastasis by Hematoxylin-eosin(HE)staining and immunohistochemistry.Results CAF and NF were successfully isolated from patients’samples.Immunofluorescence results showed thatα-smooth muscle actin(α-SMA)was significantly overexpressed in CAF,and Western blot results indicated thatα-SMA and FAP markers were significantly overexpressed in CAF.The results of foot pad tumor model indicated that,compared with the NF group,the popliteal lymph nodes in CAF group were significantly enlarged,and the difference was statistically significant(P<0.05).HE staining and immunohistochemical staining of cytokeratin 7(CK7)showed that the ratio of positive lymph nodes in CAF group was significantly increased(P<0.05),and CK7 expression was significantly increased.Conclusion By establishing a model of foot pad tumor metastasis in nude mice,it was confirmed that CAF could promote lymphatic metastasis of gallbladder cancer.