细胞外信号调节激酶反义寡核苷酸对支气管哮喘血清被动致敏的人气道平滑肌细胞的影响

目的 观察细胞外信号调节激酶(ERK)反义寡核苷酸(ODNs)对支气管哮喘(简称哮喘)血清被动致敏的人气道平滑肌细胞(HASMCs)增殖和凋亡的影响.方法 用10%哮喘患者血清被动致敏HASMCs(空白对照组),并用脂质体将反义(AODNs组)、正义(SODNs组)及错配ERKODNs(RODNs组)导入HASMCs,以10%非哮喘患者血清为对照(对照血清组).采用流式细胞仪、四甲基偶氮唑盐(MTT)法、3H-TdR掺入法及增殖细胞核抗原(PCNA)免疫荧光技术检测HASMCs的增殖,原位末端标记法和Annexin-V FITC PI双染色法检测细胞凋亡,RT-PCR和Western blot检测ERKmRNA和ERK1/2、磷酸化ERK1/2蛋白的表达.结果 用ERK ODNs干预经哮喘血清致敏的HASMCs后,AODNs组S+G2/M期细胞所占比例、吸光度(A490)值、细胞DNA合成量和PCNA蛋白表达量[分别为(14.21±1.21)%、(0.271±0.021)、(2811±182)cpm/106和(5.25±0.60)]均低于空白对照组[分别为(22.48±2.04)%、0.507±0.090、(3869±396)cpm/106和11.25±1.21](F值分别为65.594、39.676、61.111、120.321,均P<0.01),AODNs组的凋亡指数和早期凋亡细胞百分率[分别为13.96±1.72和(9.17±0.47)%]均高于空白对照组[分别为5.37±0.05和(3.26±0.04)%],差异有统计学意义(F值分别为98.181和65.444,均P<0.01),AODNs组的ERK mRNA和ERK活化率[分别为0.43±0.06和(63±6)%]均低于空白对照组[分别为0.89±0.09和(87±8)%](F值分别为78.043和87.288,均P<0.01).而正义和错配ERK ODNs没有上述作用.结论 反义ERK可通过抑制ERK mRNA表达和翻译抑制哮喘血清被动致敏的HASMCs的增殖,促进其凋亡,ERK信号通道可能对哮喘患者HASMCs增殖与凋亡具有重要的调控作用.

Effect of extracellular signal-regulated kinase antisense oligodeoxynucleotides on the proliferation of passively sensitized human airway smooth muscle cells

Objective Airway smooth muscle (ASM) cell proliferation is a key feature of airway remodeling in asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the proliferation of ASM of asthmatic rats. However, its role in the human ASMCs proliferation remains unclear. Methods HASMCs were cultured in vitro and passively sensitized with 10% serum from asthmatic patients, and non-asthmatic human serum treated HASMCs served as the control. Then, HASMCs were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides(ODN). The proliferations of HASMCs were detected by flow cytometry analysis, MTT colorimetric assay, [3H] thymidine incorporation and proliferating cell nuclear antigen ( PCNA) in immunofluorescence staining respectively. The apoptosis of HASMCs were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein and phosphorylation of ERK1/2(p-ERK1/2) protein were measured by RT-PCR and Western blotting, respectively. Results The percentage of S + G2/M phase, absorbance ( A490) value, DNA synthesis value and the expression of PCNA protein in HASMCs passively sensitized with 10% serum from asthmatic patients were (22. 48 ± 2. 04)% ,(0.507 ±0.090) ,(3869 ±396) cpm/106cells,(11. 25 ±1. 21), respectively. After treated with ERK oligodeoxynucleotides, these measurements were decreased to(14.21 ± 1. 21)% , (0. 271 ±0.021), (2811 ±182) cpm/106 cells and (5.25 ±0.60), respectively ( F = 65. 594, 39. 676,61. 111, 120. 321, respectively, P < 0. 05 ). The apoptotic index (13. 96 ± 1. 72) and the percentage of the early apoptotic cells (9. 17 ±0. 47)% in HASMCs from group AODNs were significantly increased compared to those of chronic asthma group, which were (5. 37 ± 0. 05 ), (3. 26 ± 0. 04 )% , respectively ( F = 98. 181, 65.444, respectively, P<0. 05). The expression of ERK mRNA (0.43 ±0.06) and the activation ratio of ERK (63 ±6) % in HASMCs from group AODNs were significantly decreased compared to those of chronic asthma group, which were (0. 89 ± 0. 09), (87 ± 8) % , respectively ( F = 78.043,87. 288, respectively, P < 0. 05 ). ERK antisense ODN inhibited the proliferation of HASMCs and induced the apoptosis of HASMCs, but the sense and the mismatched ones did not have these effects. Conclusions ERK antisense ODN inhibited the proliferation and increased the apoptosis in cultured HASMCs passively sensitized with 10% serum from asthmatic patients. The result suggests that ERK signaling pathway may contribute to the proliferation and apoptosis of HASMCs in asthmatic patients.