肿瘤相关细胞的诱导培养和鉴定及其对SKOV3细胞生物学行为的影响

Inductive Cultivation and Identification of Tumor-Associated Cells and Their Effects on Biological Behaviors of SKOV3 Cells

摘要: 肿瘤微环境(TME)是促进肿瘤生长的复杂系统。肿瘤微环境中的多种基质细胞以及它们分泌的细胞因子和趋化因子共同促进肿瘤细胞的增殖、迁移和侵袭。而肿瘤细胞也会分泌一些细胞因子促进肿瘤微环境中脉管系统的巩固，导致脉管系统为肿瘤细胞提供更多营养物质的同时抑制药物的运送，使其产生耐药性。本研究针对肿瘤相关细胞与肿瘤细胞间的相互作用问题，进行了体外诱导正常成纤维细胞和血管内皮细胞为肿瘤相关成纤维细胞(CAF)和肿瘤相关血管内皮细胞(TEC)，鉴定其相关性及对人卵巢癌细胞(SKOV3)生物学行为影响的研究，为肿瘤微环境多靶标协同调控的肿瘤治疗新策略奠定实验基础。实验结果表明，诱导培养后的人脐静脉血管内皮细胞(HUVEC)和成纤维细胞系(CNLMG)形态高度符合肿瘤细胞形态特征。CCK8测试结果显示，肿瘤相关细胞和肿瘤细胞呈相互促进、相辅相成的作用。ELISA和WB结果表明，肿瘤相关细胞过表达VEGF、TEM1、α-SMA和FAP标志蛋白。迁移和侵袭实验结果表明肿瘤相关细胞可以大大增强肿瘤细胞的迁移和侵袭能力。细胞周期实验反映出共培养后的SKOV3在S期的占比较大，由此可知，肿瘤相关细胞可增强SKOV3细胞的增殖能力。在荧光图像中，共培养后的SKOV3细胞吸收更少的药物并且具有更强的细胞活性，共培后的SKOV3细胞耐药性更强。研究结果表明，肿瘤细胞可以在体外诱导正常细胞成为肿瘤相关细胞，肿瘤相关细胞会影响肿瘤细胞的生物学行为。

Abstract: The tumor microenvironment (TME) is a complex system that facilitates the growth of tumors. A variety of stromal cells in the tumor microenvironment and their secreted cytokines, chemokines promote the ability of tumor proliferation, migration and invasion. The tumor cells will also secrete some cytokines to promote the consolidation of the vasculature in the tumor microenvironment, resulting in the vasculature providing more nutrients to the tumor cells while inhibiting the delivery of drugs, making them resistant. As a result, the interdependence and mutual promotion between tumor microenvironment and tumor cells greatly weaken the effect of drug treatment. This study aimed at the interaction between tumor-associated cells and tumor cells, conducted in vitro induction of normal fibroblasts and vascular endothelial cells into tumor-associated fibroblasts (CAF) and tumor-associated vascular endothelial cells (TEC), verified their correlation and the effect on the biological behavior of human ovarian cancer cells (SKOV3) the study. The experimental results show that the morphology of human umbilical vein endothelial cells (HUVEC) and fibroblast cell line (CNLMG) after induction culture is highly consistent with the morphological characteristics of tumor cells. CCK8 test results show that tumor-associated cells and tumor cells promote each other and complement each other. ELISA and WB results showed that tumor-associated cells overexpress VEGF, TEM1, α-SMA and FAP marker proteins. The results of migration and invasion experiments show that tumor-associated cells can greatly enhance tumor cell migration and invasion capabilities. The cell cycle experiment reflected that the co-cultured SKOV3 accounted for a larger proportion in the S phase, which shows that tumor-associated cells can enhance the proliferation ability of SKOV3 cells. In the fluorescence image, the co-cultured SKOV3 cells absorb less drugs and have stronger cell viability, and the co-cultured SKOV3 cells are more resistant These results indicate that tumor cells can induce normal cells to become tumor-associated cells in vitro, and tumor-associated cells can affect the biological behavior of tumor cells.