ADAM33对人气道平滑肌细胞增殖、凋亡的作用及IL-4对其表达的影响

Effects of ADAM33 on proliferation and apoptosis of human airway smooth muscle cells and effects of IL-4 on expression of ADAM33

目的探讨解整合素-金属蛋白酶33(ADAM33)对人气道平滑肌细胞(HASMCs)增殖和凋亡的作用,以及白介素-4(IL-4)对ADAM33表达的影响。方法分别以1、10、50和100 ng/mL浓度的IL-4刺激HASMCs,以未刺激细胞组作为对照,分别采用Real-Time PCR和Western blotting法检测ADAM33 mRNA和蛋白的表达。设计并合成ADAM33-siRNA,瞬时转染HASMCs,Real-Time PCR法和Western blotting法筛选出抑制率最高的siRNA,以抑制率最高的siRNA瞬时转染HASMCs;MTS和流式细胞术分别检测细胞的增殖和凋亡情况。结果当不同浓度的IL-4刺激HASMCs时,ADAM33 mRNA和蛋白的表达均呈浓度依赖性。与对照组比较,IL-4浓度为50 ng/mL组和100 ng/mL组mRNA和蛋白的相对表达量分别增加了5.94、7.6倍和3.72、4.57倍,差异均有统计学意义(P<0.05);但1 ng/mL组和10 ng/mL组无明显升高,差异均无统计学意义(P>0.05)。在3条干扰RNA中,ADAM33-siRNA-575的抑制效率最高,在mRNA水平的抑制率为81.08%。在24、48和72 h,干扰组的细胞增殖明显低于阴性对照组(P<0.05),增殖抑制率分别为15.38%、32.83%和22.78%;干扰组的细胞凋亡率为(25.88±3.37)%,明显高于阴性对照组的(8.95±0.51)%(P<0.05)。结论 ADAM33可促进HASMCs增殖,而细胞因子IL-4可促进其表达,两者可能在气道重塑中起着重要的作用。

Objective To investigate the effects of a disintegrin and metalloprotease 33 （ADAM33） on the proliferation and apoptosis of human airway smooth muscle ceils （HASMCs） and the effects of interleukin-4 （IL-4） on the expression of ADAM33. Methods The HASMCs were stimulated by IL-4 in concentrations of 1, 10, 50, and 100 ng/mL. The unstimulated HASMCs were used as controls. The expressions of ADAM33 mRNA and ADAM33 protein were detected by the Real-Time PCR and Western blotting. ADAM33-siRNA was designed, synthesized, and transiently transfected to HASMCs. siRNA with the highest inhibition rate was screened by the Real-Time PCR and Western blotting and transiendy transfected to HASMCs. The proliferation and apoptosis of cells were detected by the MTS and flow cytometry, respectively. Results The expressions of ADAM33 mRNA and ADAM33 protein were concentration-dependent for different concentrations of IL-4. Compared to the control group, the relative expressions of mRNA and protein of groups with IL-4 of 50 ng/mL and 100 ng/mL increased by 5.94 and 7.6 times, and 3.72 and 4.57 times, respectively. The differences were statistically significant （P〈0.05）. But the relative expressions of mRNA and ADAM33 protein of groups with IL-4 of 1 ng/mL and 10 ng/mL were not significant increased. The differences were not statistically significant（ P〉0.05）. For three siRNA, the inhibition rate of ADAM33-siRNA-575 was the highest. The inhibition rate was 81. 080/o at the mRNA level. The cell proliferation of the intervention group was significantly lower than that of the negative control group at 24, 48, and 72 h （P〈0.05）. The inhibition rates were 15.38%, 32.83%, and 22.780/0, respectively. The apoptotic rate of the intervention group was （25.88 ±3.37）%, which was significandy higher than that of the negative control group [（8.95 ±0.51）0/0] （P〈0.05）. Conclusion ADAM33 can promote the proliferation of airway smooth muscle cells and cytokine IL-4 can promote its expression. Both of them may play important roles in airway remodeling.