二氧化硅刺激U937细胞介导VEGF-C生成对人淋巴内皮细胞管状结构形成的影响

Effect of VEGF-C production mediated by SiO2 stimulated U937 cells on tubular structure formation of human lymphatic endothelial cells

[背景]课题组前期动物实验研究表明,二氧化硅(SiO2)诱导的矽肺大鼠早期肺组织中出现大量淋巴管生成,其对于清除肺内粉尘,缓解矽肺发病进程具有重要意义,但淋巴管生成的具体分子机制尚未明确。[目的]探究SiO2介导人单核巨噬细胞株U937产生血管内皮生长因子C(VEGF-C)对人淋巴内皮细胞(HLECs)管状结构形成的影响及作用机制。[方法]采用不同质量浓度(后称:浓度)(0、25、50、100、200 mg·L^-1)SiO2刺激U937细胞不同时间(12、24、48 h)。Western blotting检测U937细胞中VEGF-C蛋白水平,ELISA法检测细胞培养上清液中VEGF-C蛋白水平。选择VEGF-C蛋白表达水平最高的SiO2刺激浓度和时间,收集其细胞培养上清液作为条件培养基(CM)。HLECs分为阴性对照组、CM对照组、CM处理组、VEGF-C处理组。CM处理组采用SiO2刺激后的CM处理,VEGF-C处理组采用含质量浓度5000 ng·L^-1的人VEGF-C重组蛋白培养基处理,CM对照组采用未经SiO2刺激的细胞培养上清液处理,阴性对照组为常规培养。HLECs增殖能力实验,CM处理组采用体积分数为10%、50%和100%的CM处理细胞,培养24、48、72 h;迁移能力和管状结构形成能力实验均采用100%CM处理细胞,分别培养12 h和6 h。通过MTS法检测HLECs增殖能力,划痕实验和基质胶管形成实验检测细胞迁移和管状结构形成能力。[结果]与0 mg·L^-1组相比,SiO2浓度为50 mg·L^-1,刺激时间为24 h时,VEGF-C蛋白水平在U937细胞和细胞培养上清液中均最高[分别为(1.12±0.08)和(5464.00±231.21)ng·L^-1]。随着作用时间(24、48、72 h)的延长,CM浓度越大,HLECs的增殖能力越强,72 h时100%CM处理组的细胞增殖能力最强(2.00±0.13),与对照组相比差异具有统计学意义(P<0.05);72 h时VEGF-C处理组HLECs的增殖能力增强(1.67±0.10),与对照组相比差异具有统计学意义(P<0.05)。CM处理组和VEGF-C处理组中HLECs迁移面积比[(53.64±4.74)%、(56.82±5.72)%]增加,与对照组相比差异具有统计学意义(P<0.05)。CM处理组和VEGF-C处理组中HLECs的管腔数量(7.20±1.30、8.20±1.64)和管分枝数量(32.60±5.13、38.20±8.70)增加,与对照组相比差异具有统计学意义(P<0.05)。[结论]SiO2可能通过介导人单核巨噬细胞株U937产生VEGF-C,从而促进HLECs的增殖、迁移及管状结构形成。

[Background]Our preliminary animal experiment results show that silica(SiO2)induces significant lymphangiogenesis in rat lung tissues in early stage,which may be of great significance in removing lung dust and alleviating the pathogenesis of silicosis.However,the specific molecular mechanism of lymphangiogenesis has not been fully understood.[Objective]This experiment explores the effect of SiO2 stimulated human mononuclear macrophages U937 producing vascular endothelial growth factor C(VEGF-C)on the tubular structure formation of human lymphatic endothelial cells(HLECs).[Methods]U937 cells were stimulated with different concentrations of SiO2(0,25,50,100,and 200 mg·L^-1)for different time(12,24,and 48 h).Western blotting and ELISA were used to detect the VEGF-C protein level in U937 cells and in cell culture supernatant respectively.The combination of SiO2 stimulation concentration and time generating the highest VEGF-C protein expression level was selected for further experiment,and the cell culture supernatant was collected as conditioned medium(CM).HLECs were divided into four groups.The CM treatment group was treated with CM stimulated by SiO2,the VEGF-C treatment group with a medium containing 5000 ng·L^-1 human VEGF-C recombinant protein,the CM control group with cell culture supernatant without SiO2 stimulation,and the negative control group without any administration.The cell proliferation of HLECs was detected by MTS method after the cells were treated by 10%,50%,and 100%CM for 24,48,and 72 h;the cell migration and tubular structure formation were detected by scratch test and matrigel tube formation test after the cells were treated by 100%for 12 h and 6 h respectively.[Results]The SiO2 stimulation at 50 mg·L^-1 for 24 h produced the highest VEGF-C protein level in both U937 cells and cell culture supernatant[(1.12±0.08)and(5464.00±231.21)ng·L^-1 respectively]compared with the 0 mg·L^-1 group.With time extension(24,48,and 72 h),the CM concentration was higher,and the proliferation of HLECs was enhanced.The 100%CM treatment group at 72 h had the highest cell proliferation(2.00±0.13),the VEGF-C treatment group at 72 h also showed increased proliferation(1.67±0.10),and the differences were significant compared with the control group(P<0.05).The migration area ratios of HLECs in the CM treatment group and the VEGF-C treatment group[(53.64±4.74)%and(56.82±5.72)%respectively]were increased compared with the control group(P<0.05).The number of tubes(7.20±1.30,8.20±1.64)and the number of tube branches(32.60±5.13,38.20±8.70)of HLECs were increased in the CM treatment group and the VEGF-C treatment group compared with the control group(P<0.001).[Conclusion]SiO2 may promote the proliferation,migration,and tubular structure formation of HLECs by stimulating human mononuclear macrophages U937 to produce VEGF-C.