维生素D对原代培养哮喘大鼠气道平滑肌细胞增殖的影响

Effect of vitamin D on the proliferation of airway smooth muscle cells of primary culture in asthmatic rat

目的:探讨维生素D对哮喘大鼠气道平滑肌细胞(airway smooth muscle cells,ASMCs)的抗增殖机制,并证明细胞周期蛋白D1(cyclin D1)在其抗增殖调控中的作用。方法:原代培养正常对照组和哮喘组大鼠ASMCs,取第3～5代细胞用于实验。总分为以下5组:正常对照ASMCs组、哮喘ASMCs组、哮喘ASMCs+1×10-8mol/L维生素D组、哮喘ASMCs+1×10-7mol/L维生素D组、哮喘ASMCs+1×10-6mol/L维生素D组。用四甲基偶氮唑盐(MTT)法、流式细胞术检测各组ASMCs增殖活性的变化;用逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹(Western blot)检测不同组ASMCs cyclin D1 mRNA和蛋白的表达。结果:①哮喘ASMCs组G0/G1期细胞比例(80.01±2.55)%明显少于其他各组,S期(8.31±0.42)%和G2/M期(11.21±1.43)%比例增加,MTT法检测其吸光度值增高(0.78±0.04),提示ASMCs增殖明显增强。其中各值分别与正常对照ASMCs组[(90.15±3.02)%、(5.41±0.62)%、(4.38±0.56)%、0.22±0.04]相比,有显著性差异(P<0.001);同时哮喘ASMCs组与哮喘ASMCs+1×10-6mol/L维生素D组各值[(89.08±2.81)%、(5.58±0.92)%、(4.61±1.12)%、0.38±0.02]相比,其差异有统计学意义(P<0.001);与哮喘ASMCs+1×10-7/1×10-8mol/L维生素D组相比,差异也有统计学意义(P<0.01);②在cyclin D1 mRNA和蛋白表达水平上,哮喘ASMCs组与正常对照ASMCs组、哮喘ASMCs+1×10-6mol/L维生素D组分别相比较均偏高,差异显著(P<0.001);③在各不同剂量维生素D作用组,分别在细胞增殖活性检测、cyclin D1 mRNA和蛋白表达水平上,大剂量维生素D组作用均优于最小剂量组,差异有统计学意义(P<0.001),显示维生素D的抗增殖作用有明显剂量-效应相关性。结论:哮喘组大鼠ASMCs增殖明显,维生素D可以通过调节cyclinD1表达发挥其对哮喘大鼠ASMCs的抗增殖作用。

Objective：To study the anti-proliferative effect of vitamin D on the proliferation of airway smooth muscle cells（ASMCs） and prove the related mechanism of cyclin D1 and vitamin D in asthmatic rat.Methods：The ASMCs in male Sprague Dawley（SD） rats of control group and asthma group were cultured from primary generation.Cells between passage 3 and 5 were used in the study and were divided into control group,asthma group,asthma ＋ 1 × 10-6 mol/L vitamin D group,asthma ＋ 1 × 10-7 mol/L vitamin D group and asthma ＋ 1 × 10-8 mol/L vitamin D group.The proliferation of ASMCs among each group was examined by MTT c olorimetric assay and cell cycle analysis.The expression of cyclin D1 was detected by reverse transcriptase polymerase chain reaction（RT-PCR）and Western blot,respectively.Results： ①The percentage of S（8.31 ± 0.42）% and G2/M（11.21 ± 1.43）% phase,the absorbance（A）（0.78 ± 0.042） value in ASMCs from asthma group were significantly increased compared with other groups;but the percentage of G0/G1 [（80.01 ± 2.55）%] phase were significantly decreased,and compared to those（G0/G1,S and G2/M phase,A value） in ASMCs from control group [（90.15 ± 3.02）%,（5.41 ± 0.62）%,（4.38 ± 0.56）%,0.22 ± 0.04] and asthma ＋ 1 × 10-6 mol/L vitamin D group [（89.08 ± 2.81）%,（5.58 ± 0.92）%,（4.61 ± 1.12）% and 0.38 ± 0.02,P 0.001].The percentage of G0 / G1,S and G2 / M phas,A value in ASMCs from asthma ＋ 1 × 10-7 mol / L vitamin D group and asthma ＋ 1 × 10-8 mol / L vitamin D group were compared with asthma group respectively（P 0.01）;②There were significant differences in the ratios of A value of cys D1 mRNA and expression of cyclin D1 protein in ASMCs from asthma group compared with control group and asthma ＋ 1 × 10-6 mol / L vitamin D group respectively（P 0.001）;③And in the proliferation of ASMCs and the cyclin D1 mRNA and protein expression among different concentration of vitamin D groups,there were significant differences between the high and the low concentration（P 0.001）.Conclusion：ASMCs in asthmatic rats proliferate significantly and the expression of cyclin D1 increases obviously.Vitamin D can suppress the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.