p38MAPK信号传导通路调控VEGF诱导肝癌细胞黏附作用

Role of p38MAPK signal transduction pathway in adhesion of hepatocellular cancinoma cell line HepG_2 induced by vascular endothelial growth factor

目的:观察血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)通过p38信号传导通路诱导肝癌HepG2细胞转移及其对黏附作用的影响．方法:以p38MAPK信号通路特异性阻断剂SB203580预处理肝癌HepG2细胞,采用 3H-TdR掺入及鼠尾胶黏附实验测定不同浓度 VEGF对肝癌HepG2细胞同质性和异质性黏附作用,流式细胞术检测VEGF诱导肝癌细胞 CD44v6表达．结果:1 μg／L和5 μg／L VEGF诱导HepG2细胞60 min 3H-TdR掺入实验结果(dpm／min)分别为1 758．67±289．46、1 380．03±328．55; 90 min分别为3 124．30±2 262．14、2 245．60 ±273．24;10 μg／L VEGF诱导HepG2细胞60, 90,120 min 3H-TdR掺入实验分别为1 232．32 ±201．04、2 337．50±333．04、2 236.99± 237．07,显著低于对照组的2 184．49±336．03、 3 560．00±255．17、4 337．40±377．35(P<0．05 或0．01);经SB203580预处理的HepG2细胞,60, 90,120 min后的结果为2 634．23±375．21、 3 834．82±535．79、4 398．40±564．76,VEGF 诱导肝癌细胞同质性黏附作用降低．5 μg／L和 10 μg／L VEGF诱导HepG2细胞60 min鼠尾胶黏附实验吸光度A值为0．263±0．021、0．238 ±0．034,1,5和10 μg／L VEGF诱导HepG2细胞90 min A值分别为0．269±O．023、0．373± 0．083、0．393±0．081;120 min分别为0．371± 0．061、0．390±0．074、0．433±0．122,分别高于对照组的0．130±0．025、0．143±0．036、 0．210±0．028(P<0．05或0．01);经SB203580预处理的HepG2细胞,60,90,120 min鼠尾胶黏附实验吸光度A值分别为0．201±0．035、0．347± 0．112、0．479±0．217,VEGF诱导的肝癌细胞异质性黏附作用降低．5μg／L VEGF诱导肝癌细胞2 h后CD44v6表达阳性细胞数为32．6％± 4．2％,用SB203580阻断p384信号传导通路后, CD44v6阳性细胞数(4．3％±0．54％)显著下降 (P<0．01)．结论:VEGF可以通过p384信号传导通路增加肝癌细胞异质性黏附作用以及降低肝癌细胞的同质性黏附作用,促进肝癌HepG2细胞侵袭与转移．

AIM： To investigate the role of the p38MAPK signal transduction pathway in the metastasis and adhesion of hepatocellular carcinoma cell line HepG2 induced by vascular endothelial growth factor （VEGF）. METHODS： Hepatocellular cancinoma cell line HepG2 were or not pretreated with specific blocker （SB203580） of p38MAPK signal transduction pathway. ^3H-TdR infiltration and the experiment of rat tail colloid were used to measure the effects of different concentrations of VEGF on the homotypic and hetertypic adhesion in HepG2 cells. Flow cytometry was adopted to detect VEGF-induced expression of CD44v6 and by Boyden-Chamber assay was used to evaluate VEGF-induced metastasis of HepG2 cells. RESULTS： After 1 and 5 μg/L VEGF induction for 60 min, the values （dpm/min） of ^3H-TdR infiltration in HepG2 cells were 1 758.67 ± 289.46 and 1 380.03 ± 328.55; for 90 min, the values were 3 124.30 ± 2 262.14 and 2 245.60 ± 273.24, respectively. After 10 μg/L VEGF induction, the values were 1 232.32 ± 201.04, 2 337.50 ± 333.04, and 2 236.99 ± 237.07, respectively, which were dramatically lower than those in control group （P 〈 0.05 or 0.01）. However, in the HepG2 cells pretreated with SB203580, the values of ^3H- TdR infiltration after 60, 90 and 120 min were 1 232.32 ± 201.04, 2 337.50 ± 333.04, and 2 236.99 ± 237.07, respectively, and the homotypic adhesion induced by VEGF were inhibited. After 5 and 10 μg/L induction for 60 min, the optical densities （A values） of the experiment of rat tail colloid were 0.263 ± 0.021 and 0.238 ± 0.034, respectively. After 1, 5, and 10 μg/L VEGF induction for 90 min, the A values were 0.269 ± 0.023, 0.373± 0.083, and 0.393 ± 0.081, respectively; for 120 min, the A values were 0.371± 0.061, 0.390 ± 0.074, and 0.433± 0.122, respectively, in HepG2 cells, which were remarkably higher than those in control group （P 〈 0.05 or P 〈 0.01）. However, in the HepG2 cells pretreated with SB203580, the A values after 60, 90, and 120 min were 0.201 ± 0.035, 0.347± 0.112, and 0.479± 0.217, respectively, and the hetertypic adhesion induced by VEGF was restrained. After 5 μg/L VEGF induction for 2 h, the CD44v6 positive HepG2 cells counted for a percentage of 32.6% ± 4.2%, but rate of positive cell decreased to 4.3% ± 0.54% when p38MAPK signal transduction pathway was blocked （P 〈 0.01）. CONCLUSION： VEGF can promote the hetertypic adhesion and decrease the homotypic adhesion of hepatocellular carcinoma HepG2 cells by through p38MAPK signal transduction pathway, which play an important role in the invasion and metastasis of HepG2 cells.