ADAM33对人肺成纤维细胞增殖、周期、凋亡的影响及IL-4对其表达影响的研究

Effect of ADAM33 on human lung fibroblasts' s proliferation,cycle,apoptosis and the effect of IL-4 on its expression

目的探讨解整合素-金属蛋白酶33(ADAM33)对人肺成纤维细胞MRC-5增殖、周期、凋亡的影响,以及白介素-4(IL-4)对其表达的影响。方法分别以1 ng/ml、10 ng/ml、50 ng/ml、100 ng/ml浓度的IL-4刺激MRC-5,以未刺激组细胞为对照,实时荧光定量PCR法检测ADAM33 mRNA的表达情况,Western blotting法检测蛋白表达情况;设计并合成ADAM33-siRNA,瞬时转染MRC-5,MTS法检测增殖情况,流式细胞术检测细胞周期和凋亡。结果当不同浓度的IL-4刺激MRC-5时,ADAM33 mRNA和蛋白的表达均呈浓度依赖性,1 ng/ml组与0 ng/ml组相比较,差异无统计学意义(P>0.05);10 ng/ml、50ng/ml、100 ng/ml与0 ng/ml组相比较,差异有统计学意义(P<0.05);ADAM33-siRNA明显抑制了ADAM33在MRC-5中的表达;MTS结果显示,在24、48、72 h,干扰组的增殖明显低于阴性对照组,抑制率分别为27.3%、36.6%、19.2%;干扰组S期占的比例(31.69±4.14)%明显低于阴性对照组(47.19±0.99)%(P<0.05);干扰组的细胞凋亡率(28.49±8.79)%明显高于阴性对照组(8.43±5.11)%(P<0.05)。结论 ADAM33可促进人肺成纤维细胞增殖,而细胞因子IL-4可促进其表达,它们可能在气道重塑中起着重要的作用。

Objective To investigate the proliferation,cycle and apoptosis of a disintegrin and metalloprotease 33（ ADAM33） on human lung fibroblasts（ MRC-5）,and the effect of interleukin-4（ IL-4） on its expression. Methods 1 ng / ml,10 ng / ml,50 ng / ml,100 ng / ml concentrations of IL-4 stimulated MRC-5,respectively,and the unstimulated cells as the control group. real-time PCR detect the expression of ADAM33 mRNA,Western blotting assay detect its protein expression; designed and synthesized ADAM33-siRNA,transiently transfected into MRC-5,real-time quantitative PCR and Western blotting methods to detect the inhibition rate of siRNA; then,transiently transfected into MRC-5,MTS assay detected proliferation,flow cytometry detected the cycle and apoptosis. Results When different concentrations of IL-4 stimulated MRC-5,the expression of ADAM33 mRNA and protein showed a concentration-dependent manner,1 ng / ml group was compared with 0 ng / ml group,the difference was not statistically significant（P〈0. 05）,10 ng / ml,50 ng / ml and 100 ng / ml were compared with 0 ng / ml group,the difference were statistically significant（P〈0. 05）;MTS results were shown in 24 h,48 h,72 h,the proliferation of interference group was significantly lower than the negative control group,the inhibition rates were 27. 3%,36. 6%,19. 2%,respectively; The proportion of S phase in interference group was（ 31. 69 ± 4. 14） %,which was significantly lower than the negative control group（ 47. 19 ± 0. 99） %（P〈0. 05）; and the interference group apoptosis rate（ 28. 49 ± 8. 79） % was significantly higher than the negative control group（ 8. 43 ± 5. 11） %（P〈0. 05）. Conclusion ADAM33 can promote human lung fibroblasts proliferation,and cytokine IL-4 can promote its expression,they may play an important role in airway remodeling.