CAJ | 학술논문

目的探讨细胞外信号调节激酶(ERK)1/2通路在N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)抑制血小板源性生长因子(PDGF)诱导的大鼠肺成纤维细胞增殖和胶原合成中的作用.方法取新生Wistar大鼠20只,获得原代及传代培养的肺成纤维细胞.实验分为:(1)对照组(含体积分数为0.4%的胎牛血清的DMEM组);(2)PDGF组;(3)PD98059+PDGF组(25 μmol/L PD98059+10 ng/ml PDGF);(4)AcSDKP+PDGF组(1x10-8mol/L AcSDKP+10 ng/ml PDGF).采用噻唑蓝(MTT)法检测肺成纤维细胞的代谢活力,免疫细胞化学法、Western blot法检测Ⅰ、Ⅲ型胶原蛋白表达的改变;采用Western blot法检测ERK1/2及磷酸化-ERK1/2蛋白表达的改变.结果与对照组相比,PDGF组大鼠肺成纤维细胞代谢活力增强,Ⅰ、Ⅲ型胶原蛋白表达增强,磷酸化-ERK1/2蛋白表达增高,差异均有统计学意义(P<0.05).AcSDKP+PDGF组细胞代谢活力明显低于PDGF组,免疫细胞化学法检测结果显示,AcSDKP+PDGF组Ⅰ、Ⅲ型胶原蛋白表达分别为PDGF组的69.3%和67.2%.Western blot法检测结果显示,AcSDKP+PDGF组细胞内Ⅰ、Ⅲ型胶原蛋白表达分别为PDGF组的92.4%和78.0%,磷酸化-ERK1/2蛋白表达为PDGF组的83.5%,差异均有统计学意义(P<0.05).结论 ERK1/2通路在AcSDKP抑制PDGF诱导的大鼠肺成纤维细胞增殖和胶原合成中发挥了重要作用.
목적 탐토세포외신호조절격매(ERK)1/2통로재N-을선기-사안선-천문동선-뢰안선-포안산(AcSDKP)억제혈소판원성생장인자(PDGF)유도적대서폐성섬유세포증식화효원합성중적작용.방법 취신생Wistar대서20지,획득원대급전대배양적폐성섬유세포.실험분위:(1)대조조(함체적분수위0.4%적태우혈청적DMEM조);(2)PDGF조;(3)PD98059+PDGF조(25 μmol/L PD98059+10 ng/ml PDGF);(4)AcSDKP+PDGF조(1x10-8mol/L AcSDKP+10 ng/ml PDGF).채용새서람(MTT)법검측폐성섬유세포적대사활력,면역세포화학법、Western blot법검측Ⅰ、Ⅲ형효원단백표체적개변;채용Western blot법검측ERK1/2급린산화-ERK1/2단백표체적개변.결과 여대조조상비,PDGF조대서폐성섬유세포대사활력증강,Ⅰ、Ⅲ형효원단백표체증강,린산화-ERK1/2단백표체증고,차이균유통계학의의(P<0.05).AcSDKP+PDGF조세포대사활력명현저우PDGF조,면역세포화학법검측결과 현시,AcSDKP+PDGF조Ⅰ、Ⅲ형효원단백표체분별위PDGF조적69.3%화67.2%.Western blot법검측결과 현시,AcSDKP+PDGF조세포내Ⅰ、Ⅲ형효원단백표체분별위PDGF조적92.4%화78.0%,린산화-ERK1/2단백표체위PDGF조적83.5%,차이균유통계학의의(P<0.05).결론 ERK1/2통로재AcSDKP억제PDGF유도적대서폐성섬유세포증식화효원합성중발휘료중요작용.
Objective To investigate the role of extracellular signal-regulated kinase1/2 on the inhi-bition of N-aetyi-seryi-asparty-lysyl-proline (AcSDKP) on the proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by platelet-derived growth factor(PDGF). Methods Pulmonary fibroblasts were prepared from lungs of neonatal Wistar rats as described previously. Cells were divided into 4 groups : (1) control group (0.4% FBS group) ; (2) PDGF (10 ng/ml) stimulated group; (3) PD98059 +PDGF group (25 μmol/L PD98059+ 10 ng/ml PDGF); (4) AcSDKP+PDGF group (10-8 mol/L AcSDKP+ 10 ng/ml PDGF). All ex-periments were performed in the fourth passages. Metabolic activity of fibroblasts was observed by MTT, and expressions of type Ⅰ and type Ⅲ collagen were measured by immunocytochemistry and western blot. Expres-sions of phospho-ERK1/2 and ERK1/2 were detected by western blot. Results Compared with control group, exposure of pulmonary fibroblasts to lOng/ml PDGF increased cell metabolic activity, expression of type Ⅰ and type Ⅲ collagen and phosphorylation of ERK1/2.25μmol/L PD98059 and AcSDKP both could inhibit the metabolic activity of pulmonary fibroblasts, type Ⅰ and type Ⅲ collagen synthesis and phosphorylation of ERK1/ 2 induced by PDGF, with significant differences(P<0.05). AcSDKP+PDGF group compared with PDGF stimu- lated group, metabolic activity of pulmonary fibroblasts decreased to 77.4%. lmmunocytochemistry result showed that in AcSDKP+PDGF group, expressions of type Ⅰ and type Ⅲ collagen decreased to 69.3% and 67.2% compared with those of PDGF stimulated group. Western blot result showed that in AcSDKP+PDGF group, expressions of type Ⅰ and type Ⅲ collagen decreased to 92.4% and 78.0%, phospho-ERK1/2 decreased to 83.5% compared with those of PDGF stimulated group, with significant differences (P<0.05). Conclusion ERK1/2 plays an important role in the inhibition of AcSDKP on the proliferation and collagen synthesis of cul-tured rat pulmonary fibroblasts induced by PDGF.