喉肿瘤相关成纤维细胞促进原代喉鳞状细胞癌细胞体外生长  

Laryngeal cancer associated fibroblasts promote the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro

作　　者：王媚[1,2] 吴春萍[3] 曹晓娟[4] 郑文伟[4] 范国康[1] 

机构地区：[1]浙江大学医学院附属第二医院耳鼻咽喉科,杭州310009 [2]上海中医药大学附属市中医医院耳鼻咽喉科,上海200071 [3]复旦大学附属眼耳鼻喉科医院头颈外科,上海200031 [4]嘉兴学院附属第二医院耳鼻咽喉科,嘉兴314000

出　　处：《复旦学报（医学版）》2016年第2期177-182,200,共7页Fudan University Journal of Medical Sciences

摘　　要：目的验证喉肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)对原代培养的喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)细胞的体外生长是否有促进作用。方法选取20例喉癌临床标本进行原代培养,将生长出的原代喉癌细胞中的CAFs通过差速消化法去除后继续单独培养,相差显微镜观察喉癌细胞在传代培养过程中的形态学变化,流式细胞仪检测喉癌细胞在传代过程中凋亡细胞比例的变化,Western blot检测喉癌细胞在传代过程中凋亡蛋白Caspase 3表达水平的变化,以始终与CAFs共培养传代的喉癌细胞作为对照。结果与CAFs分离后喉癌原代细胞在体外传代培养过程中生长活性很快下降,多数在3代以内停止生长。相差显微镜观察到形态学上凋亡漂浮的喉癌细胞逐代增多,流式细胞仪检测到的凋亡细胞比例逐代增加,Western blot检测到的Caspase 3表达水平逐代增加;而始终与CAFs共培养生长的LSCC细胞在传代过程中生长活性却无明显下降(P<0.05)。结论喉肿瘤相关成纤维细胞对体外原代培养的LSCC细胞的生长具有明显的促进作用。Objective To explore whether the laryngeal cancer associated fibroblasts（CAFs）can promote the growth of primarily cultured laryngeal squamous cell carcinoma（LSCC）cells in vitro.Methods Twenty LSCC specimens were collected and primarily cultured.The LSCC cells growing out of the seeded tissue fragments were separated from the CAFs by differential trypsinization and subcultured continually.The inverted phase-contrast microscope was used to observe the morphological change of the cultured LSCC cells,flow cytometry was used to quantify the proportion of apoptotic cells,and Western blot was used to detect the protein level of Caspase 3.The LSCC cells cocultured with CAFs consistently serverd as controls. Results For the LSCC cells separated from CAFs,proliferation capacity decreased rapidly in the passage culture in vitro,and the most cells were terminated within the 3 generations.The apoptotic and floating LSCC cells observed by the phasecontrast microscope,the percentage of apoptotic cells quantified by the flow cytometry,and the protein level of Caspase 3 detected by Western blot increased gradually.By contrast,no significant differences of proliferation capacity of the LSCC cells cocultured with CAFs were detected during the in vitro subculture system（P 0.05）. Conclusions The laryngeal CAFs can promote the growth of primarily cultured LSCC cells in vitro.

关 键 词：肿瘤相关成纤维细胞 喉鳞状细胞癌 原代培养 凋亡 

分 类 号：R739.65[医药卫生—肿瘤]