摘要
目的原代培养食管鳞癌相关成纤维细胞(cancer-associated fibroblast,CAF)及癌旁相关成纤维细胞(peri-tumor fibroblast,PTF),并对其生物学特性及促癌作用进行初步探讨。方法对比使用组织块贴壁法和组织解离法,原代培养并分离纯化CAFs与PTFs,采用CCK-8绘制其生长曲线,免疫荧光法对其表面标志物进行鉴定;建立原代成纤维细胞与食管鳞癌细胞系KYSE150体外共培养模型,采用Transwell和划痕实验评估成纤维细胞对食管癌细胞侵袭、迁移的影响。结果组织解离法较组织块贴壁法培养成功率更高,细胞生长更快,而组织块贴壁法纯度优于组织解离法;CAFs与PTFs均为波形蛋白(Vimentin)阳性、细胞角蛋白(CK19)阴性细胞;与PTFs相比,CAFs增殖活性明显较高,α平滑肌肌动蛋白(α-SMA)高表达;在共培养模型中,CAFs对于食管鳞癌细胞系KYSE150的侵袭、迁移行为具有显著促进作用(P<0.05),而PTFs的促进作用不明显。结论通过组织块贴壁法和组织解离法成功分离培养CAFs和PTFs,CAFs特异表达α-SMA,能够显著促进食管鳞癌细胞系KYSE150的恶性生物学行为。
Objective To primary culture esophageal squamous cell carcinoma-associated fibroblast(CAF)and peri-tumor fibroblast(PTF),and to study its biological characteristics and cancer-promoting effects.Methods CAFs and PTFs were primary cultured,separated and purified with tissue block adherence method and tissue dissociation method.Method of immunofluorescence was used to identify surface markers of fibroblasts.In vitro co-culture model of fibroblasts and esophageal squamous carcinoma(ESCC)cell line KYSE150 was established to evaluate the effect of fibroblasts on the invasion and migration of esophageal cancer by methods of Transwell and scratch test.Results The primary culture of fibroblasts by tissue dissociation method had a higher survival rate and grew faster than the tissue-block adherence method,while the purity of the latter method performed better.CAFs and PTFs were all Vimentin positive and CK19 negative cells.Compared with PTFs,CAFs showed significantly higher proliferation activity and also expressedα-SMA more abundantly.In the co-culture model,CAFs could significantly promote the invasion and migration of esophageal squamous carcinoma cell line KYSE150(P<0.05),while the promotion effect of PTFs was not obvious.Conclusion CAFs and PTFs were successfully isolated and primary cultured by tissue block adherence method and tissue dissociation method.CAFs specifically expressedα-SMA,which could significantly promote the malignant biological behavior of ESCC cell line KYSE150.
作者
兰子君
郭静宜
刘轲
孔金玉
高社干
LAN Zi-jun;GUO Jing-yi;LIU Ke;KONG Jin-yu;GAO She-gan(School of Basic Medical Sciences,Henan University of Science and Technology,Luoyang,China,471023;Henan Key Laboratory of Cancer Epigenetics,The First Affiliated Hospital of Henan University of Science and Technology,Luoyang,China,471003)
出处
《食管疾病》
2021年第3期161-165,共5页
Journal of Esophageal Diseases
基金
国家自然科学基金(81972571)。
关键词
食管鳞癌
癌相关成纤维细胞
原代培养
肿瘤微环境
esophageal squamous cell carcinoma
cancer-associated fibroblasts
primary culture
tumor microenvironment
