肝窦内皮细胞与结肠癌细胞相互作用促进肝转移的影响

摘要

目的:探讨结肠癌细胞和肝窦内皮细胞共培养对结肠癌细胞功能以及肝转移能力的影响,为结肠癌肝转移机制的研究提供一种基本实验材料.方法:体外将人肝窦内皮细胞和结肠癌细胞进行共培养21轮,采用Transwell法、明胶酶谱分析、CCK-8法、集落形成实验检测共培养后结肠癌细胞SW1116P21体外侵袭、增殖、克隆形成能力;采用皮下成瘤实验以及实验性肝转移实验检测SW1116P21皮下成瘤以及肝转移的影响;Western blot的方法检测相关分子的改变.结果:共培养后的结肠癌细胞SW1116P21的细胞间界限明显.侵袭检测发现,SW1116P21细胞的侵袭能力较SW1116亲本细胞提高了2倍;明胶酶谱分析检测发现,SW1116P21分泌MMP-2/9的能力显著高于亲本细胞.皮下成瘤实验发现,SW1116P21皮下成瘤能力显著增强.实验性肝转移发现,SW1116P21肝转移能力显著高于SW1116细胞.Western blot检测发现SW1116P21细胞表达E-cadherin显著下调,vimentin表达上调.结论:结肠癌细胞和肝窦内皮细胞相互作用可促进瘤细胞侵袭、增殖、克隆形成以及体内细胞生长,更易发生肝转移,这种影响是与内皮细胞相互作用后结肠癌细胞发生EMT有关.%Objective: To investigate the effect of co-culture between colon cancer cells and human liver sinusoidal endothelial cells on metastasis of the cancer cells, and to provide a basic experimental material for studying the mechanism of liver metastasis in colon cancer.Methods: The human sinusoidal endothelial cells and colon cancer cells were co-cultured in vitro for 21 rounds.Methods including Transwell migration assay, Gelatin zymography assay, CCK-8 proliferation assay and colony formation assay were used for testing the ability of invasion, proliferation, and the colony formation of cancer cells.The subcutaneous tumor formation assay and experimental liver metastasis assay were conducted to detect the ability of tumor growth and liver metastasis.The related molecular mechanism was examined by Western blot.Results: There was a clear boundary among the colon cancer cells SW1116P21, which had been cultured with the liver sinusoidal endothelial cells for 21 rounds.Compared with the SW1116 cells, the SW1116P21 parent cells were characterized by a greater invasive ability, cell proliferation and colony formation in soft agar, with a double enhancement in the ability of the cell invasion.Gelatin zymography assay showed that the ability of the SW1116P21 cells to secrete MMP-2/9 was significantly higher than that of the parental cells.The assay of subcutaneous tumor formation showed that the ability of subcutaneous tumor formation of SW1116P21 was significantly increased.It was found in the experimental liver metastases that the SW1116P21 cells had more liver metastasis compared with the SW1116 cells.Western blot showed increased vimentin expression and decreased E-cadherin expression in the SW1116P21 cells, compared with the SW1116 cells.Conclusion: The interaction between colon cancer cells and endothelial cells can promote tumor cell invasion, proliferation and colony formation in vitro, and tumor formation and liver metastasis in vivo.The epithelial–mesenchymal transition ( EMT ) in SW1116P21 cells contributes to the change of characteristics of tumor cells.