ERK／AP-1途径在蛋白酶激活受体2促进肝癌细胞增殖中的作用  

Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists

作　　者：郑艳敏[1] 谢立群[1] 李轩[1] 赵军艳[1] 陈小义[2] 陈莉[2] 周静[1] 李飞[1] 

机构地区：[1]武警医学院附属医院消化内科,天津 300162 [2]武警医学院细胞生物学教研室

出　　处：《中华医学杂志》2009年第44期3116-3121,共6页National Medical Journal of China

摘　　要：目的探讨胰蛋白酶和蛋白酶激活受体2（PAR-2）激动剂SLIGKV．NH，对肝癌细胞增殖的影响及其细胞内信号转导机制。方法免疫荧光及逆转录（RT）-PCR法检测HepG2细胞PAR-2蛋白及mRNA的表达；用SLIGKV—NH2、胰蛋白酶、反PAR-2激动肽VKGILS—NH2及PD98059干预细胞生长。用流式细胞术检测细胞周期改变情况；噻唑蓝（MTr）法检测对细胞增殖能力的影响；RT—PCR法检测PAR-2、c-fos及增殖细胞核抗原（PCNA）mRNA表达变化；Western印迹检测c．fos和PCNA蛋白表达变化。结果肝癌HepG2细胞存在PAR-2蛋白和mRNA的表达。50μmol／ LSLIGKV—NH2、25nmol／L胰蛋白酶处理细胞后，PAR一2mRNA表达量（PAR-2／[3．肌动蛋白）分别为0．70±0．04，0．994-0．05，高于对照组（0．35-t-O．05，F=135．534，P〈0．01）。胰蛋白酶、SLIGKV—NH2组G0／G1期比例均明显低于对照组[（56．11±O．85）％、（57．85±0．46）％比（79．12±0．67）％，均P〈0．01]，s期、G：／M期细胞比例和细胞增殖指数（PI）则明显高于对照组（均P〈0．01）。胰蛋白酶、SLIGKV-NH2可以诱导肝癌HepG2细胞增殖（F=319．287，P〈0．01），上调c-fos、PCNAmRNA和蛋白的表达（均P〈0．01），且这些作用可被ERK激活性蛋白激酶（MEK）抑制剂PD98059抑制（均P〈0．01）；反PAR-2激动肽VKGILS—NH2对HepG2细胞增殖能力的影响不明显，与对照组相比差异无统计学意义（均P〉0．05）。结论肝癌HepG2细胞表达PAR-2。胰蛋白酶、SLIGKV—NH，可以通过激活PAR-2诱导HepG2细胞的增殖活性，且该过程部分由ERK／AP．1信号通路所介导。Objective To investigate the expression of protease activated receptor-2 （PAR-2） in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH2 upon the proliferation of hepatoma cells and its intracellular signaling mechanism. Methods PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH2, trypsin, reverse PAR-2 agonist peptide VKGILS-NH2 or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by M＇[T. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. Results PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression （PAR-2/[3-actin） were 0.70 ±0. 04 and 0. 99 ± 0.05 respectively in cells treated with trypsin and SLIGKV-NH2. They were both significantly higher than that in the control group （0. 35 ± 0. 05, F = 135. 534, P 〈 0. 01 ）. Percent G0/G1 phase of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly lower than those in the control group [ （56. 11 ±0. 85） %, （57.85 ± 0.46） % vs （79. 12 ± 0. 67 ） %, both P 〈 0. 01 ] Percent S phase, G2/M phase and proliferation index （PI） of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated（P 〈 0.01 ）. The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH2 were significantly blocked by pretreatment with PD98059 （ P 〈 0. 01 ）. There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH2 and control group （P 〉 0. 05）. Conclusion PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH2 promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

关 键 词：受体 PAR-2 胰蛋白酶 肝肿瘤 细胞增殖 

分 类 号：R686[医药卫生—骨科学]