食道鳞状上皮细胞癌转移体外细胞模型的建立

The development of a new in vitro cell model for studying ESCC metastasis

目的建立一个适宜的食道鳞状上皮细胞癌(ESCC)体外转移研究模型。方法将ESCC细胞系HK2置于未处理细胞培养皿中培养、传代,筛选出具有非锚定依赖生长能力的细胞株。以体外侵袭实验,软琼脂克隆形成实验比较、验证筛选细胞系与母代细胞的侵袭能力和非锚定依赖生长能力。利用比较蛋白质学的方法分析筛选细胞与母代细胞间的蛋白表达图谱差异。结果成功筛选出具有较高转移能力并可传代培养的HK2-AD细胞株。HK2和HK2-AD细胞侵袭至小室膜反侧的细胞数分别为(16.7±2.5)和(28.7±7.4),软琼脂集落生成实验表明,HK2和HK2-AD细胞形成的集落数分别为(16.8±3.2)和(25±5.1),差异均有统计学意义(P<0.05)。HK2-AD细胞中一些与细胞运动、细胞无氧代谢及抑制凋亡相关的蛋白质分子表达明显上调。结论利用肿瘤细胞的非锚定依赖生长特性建立了一个高转移能力、呈三维生长的HK2-AD细胞株,为ESCC肿瘤转移机制研究和药物筛选提供了一个有价值且易于操作的食道癌转移体外细胞模型。

Objective To develop an in vitro cell model for studying the molecular basis of metastatic process in esophageal squamous cell carcinoma （ESCC）. Methods The ESCC cell line HK2 was cultured on non-adherent surface vessel, and the cell subline HK2-AD with a selective survival advantage in non adherent conditions were developed. The invasive and anchorage independent growth abilities of HK2 and HK2-AD were determined by in vitro invasion assay and colony formation assay. The proteomic pattems of HK2 and HK2-AD were analyzed with 2DE coupled with MS/MS. Results The HK2-AD cell line was successfully established. Compared to parental HK2 cells, HK2-AD cells have increased invasive and anchorage-independent growth abilities. The cells that invaded into the lower chamber and the colony formed in soft agar were（16.7±2.5） vs.（28.7±7.4） and （16.8±3.2） vs. （25±5.1） for HK2 and HK2-AD cells respectively. The differences were statistical significance （P〈0.05）. In addition, the expression of some proteins that involved in cell motility, glycolysis, and cell protection were up-regulated in HK2-AD cells. Conclusion An in vitro 3D cell model of metastasis was established based on anchorage-independent growth ability,which is valuable and easy to operate for further studies on ESCC metastatic process and drug development.