西地那非对人淋巴管畸形内皮细胞增殖能力的影响

Effect of sildenafil on proliferation of human lymphatic malformations endothelial cells

目的 :探讨西地那非对体外培养的人淋巴管畸形内皮细胞(human lymphatic malformations endothelial cells,HLMECs)增殖的影响及可能机制。方法 :用组织块培养法对人淋巴管畸形内皮细胞进行体外培养;免疫细胞化学行CD-31、LYVE-1染色鉴定;采用第3代细胞进行后续研究,不同浓度的西地那非(1、2、5、10μmol/L)作用后,应用倒置显微镜观察HLMECs形态的变化,MTT比色法和Ed U染色法检测对细胞增殖的影响,Western免疫印迹检测血管内皮细胞生长因子C(VEGFC)及其受体VEGFR-3表达的变化。采用SPSS 21.0软件包对数据进行独立样本t检验、单因素方差分析和LSD检验。结果:组织块培养法所获细胞染色显示CD31及LYVE-1呈阳性;西地那非作用后细胞形态改变,密度降低;MTT及Ed U染色显示,西地那非作用浓度在2μmol/L时细胞增殖受到明显抑制(P<0.05);Western免疫印迹显示,VEGF-C及VEGFR-3表达水平显著下调(P<0.05)。结论 :西地那非对体外培养的人淋巴管畸形内皮细胞具有增殖抑制作用,其机制可能与调节VEGFC/VEGFR-3的表达有关。

PURPOSE： To explore the effect and mechanism of sildenafil on proliferation of human lymphatic malformations endothelial cells （HLMECs） in vitro. METHODS： Tissue block culture method was used to culture HLMECs in vitro, which were identified by immunocytochemistry （CD31 and LYVE-1）. Subsequent studies were performed using third-generation cells, the cells were treated with sildenafil at the concentration of 1, 2, 5 and 10 txmol/L, respectively, cell morphology was observed under an inverted microscope; cell proliferation activity was detected by MTI＂ and EDU staining, and the expression level of vascular endothelium growth factor C （VEGFC） and its receptor VEGFR-3 was detected by Western blot. Independent-samples t test and ANOVA （LSD method） were performed using SPSS 21.0 software package for statistical analysis. RESULTS： Cells cultured by tissue block method revealed both positive CD31 and LYVE-1 staining. After treated with sildenafil, the cell morphology was changed and the density of the cells was decreased. MTT and EDU staining reflected that the cell proliferation was significantly inhibited by sildenafil at the concentration of 2 Ixmol/L （P〈0.05）. There was also decreased level of VEGFC and VEGFR-3, detected by Western blot. CONCLUSIONS： Sildenafil could inhibit the proliferation of HLMECs in vitro. The mechanism might be relate to the regulation of VEGFC/VEGFR-3.