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
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常州市应用基础研究计划项目(CJ20112011);南京医科大学科技发展基金重点项目(2011NJMU225)


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

    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 × 10-8 mol/L维生素D组-哮喘ASMCs + 1 × 10-7 mol/L维生素D组-哮喘ASMCs + 1 × 10-6 mol/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 × 10-6 mol/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 × 10-8 mol/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 × 10-6 mol/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。

    Abstract:

    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 colorimetric 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.

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曾幸坤,郭红荣,俞小卫,刘 茗.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[J].南京医科大学学报(自然科学版),2013,(8):1070-1076

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  • 收稿日期:2013-02-17
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  • 在线发布日期: 2013-07-30
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