舒尼替尼耐药内皮细胞HMEC-su与EndMT的关系及其耐药特性

目的：研究舒尼替尼（sunitinib）耐药内皮细胞HMEC-su细胞的耐药特性，并对其与内皮间质转化（EndMT）之间的关系进行初步探讨。方法：结晶紫染色法观察HMEC-su细胞的形态学变化；MTT法检测HMEC-su细胞对阿霉素（DOX）的敏感性，并计算耐药指数（resistance index，RI）；罗丹明123（rhodamine 123，Rho 123）外排实验评价HMEC-su细胞的耐药性；流式细胞仪检测细胞周期分布；划痕修复实验评价细胞的迁移能力；Western blot实验检测HMEC-1和HMEC-su细胞中EndMT相关蛋白血管内皮细胞钙黏素（VE-cadherin）和α-平滑肌肌动蛋白（α-SMA）的表达差异。结果：与HMEC-1细胞相比，HMEC-su细胞的形态发生明显变化，增殖速度减慢；HMEC-su细胞对DOX产生交叉耐药，RI为2.15（P<0.01）；Rho123蓄积实验显示，与HMEC-1细胞相比，HMEC-su细胞内Rho123荧光强度明显降低（P<0.01），给予维拉帕米（verapamil，VRP）处理1 h后，HMEC-su细胞中Rho123荧光强度明显升高（P<0.01）；与HMEC-1细胞相比，HMEC-su细胞G₀/G₁期的细胞比例显著降低（P<0.01），然而S期和G₂/M期显著增加（P<0.01，P<0.05）；划痕修复实验表明HMEC-su细胞的迁移能力明显高于HMEC-1细胞（P<0.01）；Western blot实验表面，与HMEC-1细胞比较，HMEC-su细胞中VE-cadherin表达显著降低（P<0.01），α-SMA蛋白表达显著增加（P<0.05）。结论：HMEC-su细胞对DOX产生了交叉耐药，其细胞形态和周期分布存在着明显变化，初步确定EndMT与HMEC-su细胞耐药性的产生有关。

Involvement of EndMT in Sunitinib-resistance endothelial cell (HMEC-su) and its biological characteristics

OBJECTIVE To investigate the drug resistance characteristics of sunitinib resistant endothelial cells (HMEC-su cells) and its relationship with endothelial mesenchymal transition (EndMT). METHODS The morphological changes of HMEC-su cells were observed by crystal violet staining; the sensitivity of HMEC-su cells to doxorubicin (DOX) was detected by MTT assay, and the resistance index (RI) was calculated; the efflux assay of rhodamine 123 (Rho 123) was used to evaluate the drug resistance of HMEC-su cells; the cell cycle distribution was measured by flow cytometry; the migration ability of cells was evaluated by scratch repair assay; and the expression differences of EndMT-related proteins vascular endothelial cell cadherin (VE-cadherin) and α-smooth muscle actin (α-SMA) between HMEC-1 and HMEC-su cells were detected by Western blot assay. RESULTS Compared with HMEC-1 cell, HMEC-su cell had remarkable morphological changes and decreased proliferation ability. HMEC-su cells developed cross-resistance to DOX with RI of 2.15 (P<0.01); Rho123 accumulation assay showed that the intracellular Rho123 fluorescence intensity was significantly lower in HMEC-su cells compared with HMEC-1 cells (P<0.01), and the Rho123 fluorescence intensity was significantly higher in HMEC-su cells after treatment with verapamil (VRP) for 1 h (P<0.01); compared with HMEC-1 cells, the proportion of cells in G₀/G₁ phase of HMEC-su cells was significantly lower (P<0.01), however, the migration ability of HMEC-su cells was significantly higher than that of HMEC-1 cells in S and G₂/M phases (P<0.01, P<0.05); Western blot analysis showed that the migration ability of HMEC-su cells was significantly higher than that of HMEC-1 cells (P<0.01); VE-cadherin expression was significantly decreased (P<0.01) and α-SMA protein expression was significantly increased (P<0.05) in HMEC-su cells. CONCLUSION HMEC-su cells are cross-resistant to DOX, and there are significant changes in cell morphology and cell cycle distribution. It is preliminarily confirmed that EndMT is related to the development of drug resistance in HMEC-su cells.