人气道平滑肌细胞体外培养与被动致敏后的生物学特征

Biological characteristics of human airway smooth muscle cells in vitro cultured and passively sensitized.

目的研究特应血清被动致敏的人气道平滑肌细胞增殖、DNA合成、蛋白质合成等生物学特征.方法将正常人气道平滑肌细胞体外培养,用来源于2002～2003年在上海二医大附属瑞金医院就诊的哮喘患儿的特应血清(总IgE>106IU/L,特异性IgE≥Ⅲ级)对平滑肌细胞刺激、被动致敏,绘制被动致敏后的细胞生长曲线,比色法测定蛋白质合成量,3H-TdR结合试验测定DNA合成.结果特应血清被动致敏刺激的平滑肌细胞于培养的72h,96h,120h,细胞数分别为(5.21±0.87)×104/孔,(6.43±0.57)×104/孔,(7.25±0.65)×104/孔,同样条件下非特应血清刺激组细胞数分别为(3.78±0.50)×104/孔,(4.80±0.49)×104/孔,(5.55±0.77)×104/孔,两组在72h,96h,120h差异有显著性意义(P<0.01);特应血清致敏的平滑肌细胞蛋白质合成量为(3.81±0.52)mg/L,非致敏平滑肌细胞蛋白质合成量为(1.90±0.35)mg/L,两组相比差异有显著性(P<0.01),3H-TdR的结合力两组差异也有显著性意义[(196.4±45.2)cpm/孔和(127.2±33.3)cpm/孔,P<0.01].结论特应血清致敏对平滑肌细胞分裂和蛋白质合成有显著促进作用,过度增殖的平滑肌细胞使气道平滑肌总量增加,是发生气道重建的重要结构基础.

Objective To explore the biological characteristics of passively sensitized airway smooth muscle（ ASM ） cells in hyperplasia,protein and DNA synthesis abilities. Methods ASM cells derived from human bronchus were in vitro cultured and passive sensitized with atopic serum, which was collected from asthmatic individuals with serum total IgE 〉 10^6 IU/L, specific IgE ≥ Ⅲ. Cell counting by cell growth curve,protein synthesis by Bio-rad DC protein assay and ^3 H-TdR incorporation assays were performed, Results Passively sensitized with atopic serum, ASM cells were more active in proliferation, protein synthesis and DNA synthesis compared with non-atopic serum sensitized ASM. In details, cellular proliferation was counted after culture of 72h ,96h and 12Oh,which were （5.21 ± 0. 87） × 10^4/well, （6.43 ± 0. 57 ） ×10^4/well and （7. 25 ± 0. 65） ×10^4/well respectively in passively sensitized airway smooth muscle cells and （3.78 ± 0. 50 ） ×10^4/well, （4. 80 ± 0.49） × 10^4/well, （5.55 ± 0. 77） ×10^4/well in non-passively sensitized airway smooth muscle cells, showing significant difference（ P 〈0. 01）. Meanwhile,passively sensitized ASM cells synthesis produced more proteins than non-sensitized ASM （3.81 ± 0. 52mg/L vs 1.90 ± 0. 35mg/L, P 〈 0. 01 ） ,incorporation of ^3 H-TdR also had significant difference between two groups （ 196. 4 ± 45.2cpm/well vs 127.2 ± 33.3cpm/well, P 〈 0. 01 ）. Conclusion Atopic serum has synergetic effects on promoting ASM cells proliferation and protein synthesis. Hyperproliferation of ASM cells increase the ASM mass,which is an important change in later asthma airway remodeling.