ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡

 为了了解ERK信号通道对正常大鼠气道平滑肌细胞(airway smooth muscle 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procaspase-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Ａ_４９０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Ａ_４９０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procaspase-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.

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

The regulative role of extracellular signal-regulated kinase(ERK)signaling pathway on proliferation and apoptosis in rats' airway smooth muscle cells(ASMCs)was investigated.Primary cultures of ASMCs were established and cells between passages 4 and 7 were used for experiments.ASMCs were U-eated 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 eolorimetric assay and 3H] thymidine incorporation.Apoptosis of ASMCs were detected by Hoechst staining and Annexin-V FITC PI donble staining.The levels of ERK1/2,phosphorylated forms of ERK1/2(p-ERK1/2) and proeaspase-3 protein were detected by Western blotting.The expressions of ERK mRNA and ERK protein were obviously observed in ASMCs. Compared with control,the absorbanee(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 cells 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 c,ontrol,the A490 value and DNA synthesis index were significantly increased(P＜0.05)in EGF-treated cells.Apoptotic index and the percentage of the early apoptotic cells 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.