黑素瘤细胞中诱导型一氧化氮合酶信号调控机制及其促淋巴内皮细胞增殖

Study of induced nitric oxide enzymeregulation mechanism in melanoma cells and its effect on lymphatic endothelial cell proliferation

目的探讨B16黑素瘤细胞中诱导型一氧化氮合酶（iNOS）信号调控机制，以及该信号通路对淋巴内皮细胞（LEC）增殖的影响。方法将B16细胞iNOS信号调控实验分为4组：空白对照组、血管内皮生长因子-C（VECF-C）处理组、VEGF-C＋iNOS拮抗剂氨基胍（Ag）组、VEGF-C＋VEGF受体．3（VEGFR-3）拮抗剂MAZ-51组。培养48h后用实时定量聚合酶链反应、Western blot、硝酸还原酶法检测细胞中iNOS的mRNA、蛋白的表达水平及一氧化氮（NO）的生成量。LEC增殖实验中对小鼠LEC及B16细胞共培养，分为6组：A组（单纯LEC）、B组（LEC＋VEGF-C）、C组（LEC＋B16）、D组（LEC＋B16＋VEGF-C）、E组（LEC＋B16＋VEGF-C＋Ag）、F组（LEC＋B16＋VEGF-C＋MAZ-51）。采用5-乙炔基-2’脱氧尿嘧啶核苷（EdU）法检测LEC增殖。结果iNOS信号调控实验中，VEGF-C组中B16细胞iNOSmRNA及蛋白的表达水平较空白对照组均显著提高（1．10±0．03比0．69±0．02，P=0．008），而加有舷或MAZ-51组中iNOSmRNA及蛋白的表达水平不仅低于VEGF－C组（0．35±0．03比1．10±0．03，P=0．005；0．42±0．02比1．10±0．03，P=0．004），且低于空白对照组（0．35±0．03比0．69±0．02，P=0．028；0．42±0．02比0．69±0．02，P=0．025）。对NO检测后发现VEGF-C组中NO的吸光度（A）值为0．25±0．03，明显高于空白对照组（0．19±0．03，P=0．008），且显著高于加有Ag（0．07±0．02，P=0．017）或MAZ-51（0．07±0．02，P=0．015）组。对小鼠LEC增殖水平结果显示：各组增殖率分别为（0．45±0．02）％、（0．47±0．03）％、（0．48±0．05）％、（0．51±0．02）％、（0．24±0．01）％和（0．28±0．03）％，与空白对照组A组比较，B、C、D组中LEC的增殖水平均显著升高（P=0．029，P=0．024，P=0．009），而E、F组显著降低（P=0．006，P=0．005）。结论抑制B16细胞中VEGF-C／VEGFR-3信号通路能够减少iNOS的表达，从而进一步减少LEC的增殖。

Objective To research induced nitric oxide enzyme （iNOS） signal regulation mecha- nism in B16 melanoma cells, and the effect on lymphatic endothelial cell （LEC） proliferation. Methods In the iNOS signal regulation experiment, the cultivation of B16 cells were divided into 4 groups： blank control group, Vascular endothelial growih factor - C （VEGF - C） group, VEGF - C ＋ iNOS antagonist aminoguanidine （Ag） group, VEGF - C ＋ VEGF receptors （ VEGFR - 3） antagonist MAZ - 51 group. We used the real - time quantitative polymerase chain reaction, Western blot test and nitrate reduction method to detect the level of iNOS mRNA, protein and nitric oxide （NO） in B16 cellsfor each grouprespectively after co-cultured 48 h. In LEC proliferation study, B16 cellswere co- cultured with LEC. The sampleswere divided into 6 groups. Group A （ LEC ） ; GroupB （ LEC ＋ VEGF - C ） ; Group C （ LEC ＋ B16 ） ; Group D （LEC ＋B16 ＋VEGF -C） ; Group E （LEC ＋B16 ＋VEGF -C ＋Ag） ; Group F （LEC ＋B16 ＋VEGF -C ＋ MAZ -51 ）. EDU method was used to detect LEC proliferation. Results In iNOS signal regulation experiment： as compared to blank control group, the iNOS mRNA and protein expression level in B16 cells were significantly improved in group VEGF - C （ 1.10 ± 0. 03 vs. 0. 69 ± 0. 02, P = 0. 008 ）. While the group with Ag or MAZ -51, the level of iNOS mRNA and protein expression was not only lower than VEGF - C group （0. 35 ± 0. 03 vs. 1.10 ±0.03, P = 0. 005 ; 0. 42± 0. 02 vs. 1.10 ±0. 03, P = 0. 004）, but also lower than the blank control group （ 0. 35 ＋ 0. 03 vs. 0. 69± 0. 02, P = 0. 028 ; 0. 42 ±0. 02 vs. 0. 69 ±0.02, P = 0. 025 ）. The optical density of NO in VEGF - C group was 0. 25 ±0. 03, which was significantly higher than the blank control group （0. 19 ± 0. 03, P = 0. 008 ）, and significantly higher than the group withAg （0.07 20.02, P=0.017） orMAZ-51 （0.07 20.02, P=0. 015）. The LEC proliferationex- periment showed ： The proliferative rates were （ 0. 45±0. 02 ） %, （ 0.47 ±0. 03 ） % , （ 0. 48 ± 0. 05 ） % , （0. 51 ±0.02） % , （0. 24 ± 0. 01 ） % , and （0. 28 ± 0. 03 ） % respectively. Compared with the blank control group （A）, the level of LEC proliferation in group B, C, and D were all significantly improved（P = 0. 029, P = 0. 024, P = 0. 009）. In Group E andF were obviously lowered （ P = 0. 006, P = 0. 005 ）. Conclusion To inhibit the VEGF - C/VEGFR - 3 signaling pathways in B16 cells can reduce the expression of iNOS, and further reducing the proliferation of LEC.