ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡(英文)

Regulation of Cell Proliferation and Apoptosis by ERK Signaling Pathway in Airway Smooth Muscle Cells of Rats

为了了解ERK信号通道对正常大鼠气道平滑肌细胞（aimay smooth nilscle cells，ASMCs）增殖与凋亡的调控．通过对正常大鼠ASMCs原代培养，4～7代用于实验，以ERK激动剂表皮生长因子（EGF）和抑制剂PD98059干预ASMCs生长，采用RT-PCR和免疫荧光染色观察ASMCs上ERK mRNA和蛋白的表达，MTT法、^3H-TdR掺入法检测ASMC增殖，Hoechst染色和Annexin-Ⅴ FITC PI双染色法检测细胞凋亡，Western免疫印迹检测ERK1／2、磷酸化ERK1／2和proeaspase-3蛋白的表达。结果发现ASMCs上存在ERK mRNA和蛋白的表达，与空白对照组比较，PD98059干预后ASMCs的A490值和细胞DNA合成量均减少（P〈0．05），细胞凋亡指数、早期凋亡细胞百分率均增高（P〈0．05），ERK1／2、磷酸化ERK1／2表达和ERK活化率均降低，procaspase-3蛋白的表达增高．EGF干预后ASMCs的A490值和细胞DNA合成量均增高（P〈0．05），细胞凋亡指数、早期凋亡细胞百分率均下降（P〈0.05），ERK1／2、磷酸化ERK1／2表达和ERK活化率均增高，procaspase-3蛋白的表达降低．P＋E组无明显差异（P〉0．05）．ERK信号通道参与大鼠ASMCs增殖和凋亡的调控，ERK对大鼠ASMCs凋亡的调控与proeaspase-3蛋白有关，这一发现将有助于对哮喘ASMCs异常增殖调控机制的深入研究．

The regulative role of extracellular sigual-regulated kinase （ERK） signaling and apoptosis in rats＇ airway smooth muscle cells （ASMCs） was investigated Primary pathway on proliferation cultures of ASMCs were established and cells between passages 4 and 7 were used for experiments. ASMCs were treated with ERK activator epidermal growth factor （EGF） and inhibitor PD98059. The expressions of ERK mRNA and protein were detected by RT-PCR and immunofluorescence staining. Proliferation of ASMCs were detected by MTT colorimetric assay and [^3H ] thymidine incorporation. Apoptosis of ASMCs were detected by Hoechst staining and Annexin-V FITC PI double staining. The levels of ERK1/2, phosphorylated forms of ERK1/2（p-ERK1/2） and procaspase-3 protein were detected by Western blotting. The expressions of ERK mRNA and ERK protein were obviously observed in ASMCs. Compared with control, the absorbance （A490） value and DNA synthesis index of PD-treated ASMCs were significantly decreased （P 〈 0.05）. The apoptotic index and the percentage of the early apoptotic ceils were significantly increased （P 〈 0.05）. The expressions of ERK1/2 and pERK1/ 2 protein were significantly down-regulated. The expressions of procaspase-3 protein was significantly increased. Compared with control, the A490 value and DNA synthesis index were significantly increased （ P 〈 0.05） in EGF-treated ceils. Apoptotic index and the percentage of the early apoptotic ceils were significantly decreased （P 〈 0.05 ）. The levels of ERK1/2 and pERK1/2 protein were significantly increased, and the levels of procaspase-3 protein were significantly decreased. There were no significant differences between control and P ＋ E group（ P 〉 0.05）. ERK signaling pathway may play an important role in regulating ASMCs proliferation and apoptosis and ERK regulating the apoptosis of ASMCs possibly relates to the expressions of procaspase-3 protein. The finding will help to understand the mechanisms of asthmatic ASMCs involved in abnormal proliferation.