丝裂原活化蛋白激酶信号通路在肝细胞生长因子促进人结肠癌细胞增殖移行中的作用

Role of mitogen-activated protein kinase signaling pathway in proliferation and migration of SW620 activated by hepatocyte growth factor

目的观察丝裂原活化蛋白激酶（MAPK）信号通路在肝细胞生长因子（HGF）促进人结肠癌细胞SW620增殖、移行中的作用。方法在细胞培养液中加入MAPK通路中丝裂原细胞外激酶（MEKl／2）的拮抗剂U0126（终质量浓度分别为0．5、1．0、2．0、4．0、8．0μmol／L），之后加入终质量浓度为20μg/L的HGF。用噻唑蓝（MTT）比色法检测SW620细胞增殖，流式细胞术检测细胞周期，划痕试验检测移行。二甲基亚砜（DMSO）组加终质量浓度为0．1％体积浓度的DMSO，加入同体积的培养液作为对照组。结果（1）U0126与20μg/LHGF共同作用SW620细胞48h后，低浓度（0．5、1．0、2．0μmol／L）U0126组、DMSO组与对照组之间比较差异无统计学意义（P〉0．05），当U0126浓度≥4．0μmol／L时能抑制HGF促SW620增殖，但未表现出浓度依赖性。4．0、8．0μmol／LU0126组增殖抑制率分别为24．6％和28．4％（P〈0．05）。（2）2．0、4．0、8．0μmoL／LU0126组G1期细胞数增多，而S期细胞数减少，3组的增殖指数（32．32±6．64、23．87±4．88、20．28±5．22）均低于对照组，P〈0．05），且4．0、8．0grmol／LU0126组与2．0μmol／L组差异有统计学意义（P〈0．05）。（3）加入U012624h后，仅8．01．μmol／L组SW620细胞移行受到明显抑制，抑制率为60．9％（P〈0．05），DMSO组、4．0μmol／LU0126组与对照组移行距离差异无统计学意义（P〉0．05）；48、72h后，8．0μmol．／L和4．0μmol／LU0126组细胞移行均受到抑制（P〈0．05），48h抑制率分别为46．5％、61．8％．72h抑制率分别为44．2％、54．3％，但8．0μmol／L组与4μmol／L组细胞移行距离差异无统计学意义（P〉0．05）。结论MAPK信号通路参与了HGF促人结肠癌细胞SW620增殖和移行的作用。

Objective To investigate the effects of mitogen-activated protein kinase （MAPK） signaling pathway in proliferation and invasion of SW620 cells that were activated by hepatocyte growth factor （HGF）. Methods The mitogen extracellular kinase （MEK） 1/2 inhibitor U0126 （0. 5, 1.0, 2. 0, 4. 0, and 8.0 μmol/L） was used to block the MAPK pathway, and then, HGF （20 μg/L） was added to the culture medium. The proliferation of SW620 cells that were activated by HGF was assessed by methyl thiazol tet- razolium （MTT） assay. The cell cycle and apoptosis were examined by flow eytometry, and the migration was detected by Scratch test. Results （ 1 ） After treatment of SW620 cells with U0126 （ concentrations of 0. 5, 1.0, 2. 0 μmol/L, respectively） combined with HGF （20 mg/L） for 48 h, there was no significant difference in proliferation among U0126 group, DMSO group and control group. The proliferation of SW620 cells activated by HGF was inhibited only in 4. 0 μmol/L and 8.0 μmol/L U0126 groups with the inhibition ratio being 24. 6% and 28.4% （P 〈 0. 05）, respectively. U0126 inhibited the proliferation of SW620 cells that activated by HGF in a concentration-independent manner; （2） The number of cells in G1 phase was increased and that in S phase was decreased in U0126 （2. 0, 4. 0, and 8.0μmol/L） -treated groups com- pared to the control group （P 〈0. 05）. Proliferation index in U0126 （2. 0, 4. 0, and 8.0 μmol/L）-treated groups was 32. 32 ±6. 64, 23.87± 4. 88, and 20. 28± 5.22, respectively, which was lower than that inthe control group （P 〈0. 05） ; （3） Only the 8. 0 μmoL/L but not 4.0 p, mol/L U0126 group showed that the migration of SW620 cells that activated by 20 μg/L of HGF at 24 h was significantly inhibited. The mi- gration rate in 8.0 μmol/L U0126 group was 60. 9% （P 〈0. 05）. However, the migration of SW620 cells at 48 and 72 h was significantly inhibited in both 8.0 or 4. 0 μmol/L U0126 group （ P 〈 0. 05 ）, with the migration rate being 46. 5% and 61.8%, and 44. 2% and 54. 3%, respectively. Conclusion MAPK sig- naling pathway involves in the proliferation and migration of SW620 cells activated by HGF.