二氧化硅刺激肺成纤维细胞间隙连接功能下调的研究

Study on silicon dioxide induced downregulation of gap junctional intercellular communication of the pulmonary fibroblasts

目的 探索矽肺发病过程中二氧化硅 (SiO2 )刺激肺纤维化发生与肺成纤维细胞间隙连接通讯功能 (GJIC)下调的关系。方法 不同剂量的SiO2 刺激SD大鼠肺泡巨噬细胞 (PAM)及具有肺泡巨噬细胞特性的、经 12 O 四葵酰基 佛波 13 乙酸酯 (佛波酯 ,PMA或TPA)诱导分化的人血单核细胞株(pTHP 1)培养上清液 ,作用于中国仓鼠肺成纤维 (CHL)细胞。用四唑盐 (MTT)法测定CHL细胞增殖(A570nm) ;用激光扫描共聚焦显微镜 ,按荧光光漂白后再恢复 (FRAP)技术测定CHL细胞GJIC功能 [荧光传递速率K ,(× 10 -3/s) ]。结果 SiO2 刺激的PAM上清液和pTHP 1上清液均能诱导CHL细胞增殖(PAM :F =3.2 0 5 ,P <0 .0 5 ;pTHP 1:F =13.779,P <0 .0 1)及抑制GJIC功能 (PAM :F =19.948,P <0 .0 1;pTHP 1:F =9.36 5 ,P <0 .0 1) ,并随SiO2 浓度增加 (5 0、10 0、2 0 0和 5 0 0 μg/ml) ,细胞增殖能力渐强 ,呈剂量-效应关系 (PAM :r=- 0 .80 3,P <0 .0 5 ;pTHP 1:r =- 0 .914,P <0 .0 1)。PAM和pTHP 1细胞上清液可以增强CHL细胞的GJIC功能 (K均数分别为 2 1.2 4× 10 -3/s、18.92× 10 -3/s)和抑制细胞增殖 (A570nm均数分别为 0 .5 0 6、0 .2 18) ,和空白对照 (7.81× 10 -3/s、7.81× 10 -3/s和 0 .5 39、0 .388)相比 。

Objective To explore the relationships between pulmonary fibrosis and downregulation of gap junctional intercellular communication(GJIC) of the fibroblasts after stimulation by SiO 2. Methods The pulmonary alveolar macrophage(PAM) and the phorbol 12 myristate 13 acetate(PMA or TPA) primed THP 1(pTHP 1),a monocyte like cell line with the properties of PAM,were incubated in the serum free RPMI?1640 containing SiO 2 at various concentrations.The proliferation of Chinese hamster lung(CHL) fibroblast induced by cultured PAM or pTHP supernatant was detected by using MTT assay(to show as the absorbency, A 570 nm ),and GJIC between those cells was measured by using the fluorescence redistribution after photobleaching(FRAP) assay(to show as the transfer rate of the fluorescence, K ×10 -3 /s) performed with a laser scanning confocal microscope(LSCM,Carl Zeiss LSM 510,release 2.01). Results Both SiO 2 exposed PAM and pTHP 1 supernatants could induce the proliferation(PAM: F =3.205, P <0.05;pTHP 1: F =13.779, P < 0.01) and the downregulation of GJIC(PAM: F =19.948, P < 0.01; pTHP 1: F =9.365, P < 0.01) of the CHL cells.In the range of 0,50,100,200 and 500 μg/ml SiO 2 concentrations,the proliferation( A 570 nm values) and GJIC(the transfer rate, K )were fitted well in the dose effect relationship(PAM: r =-0.803, P <0.05;pTHP 1: r =-0.914, P < 0.01). Compared with the blank control,both PAM and pTHP 1 supernatants could upregulate GJIC( K :21.24×10 -3 /s, 18.92×10 -3 /s vs. 7.81×10 -3 /s,7.81×10 -3 /s respectively, P < 0.05) and inhibit the proliferation of CHL cell( A 570 nm : 0.506, 0.218 vs. 0.539,0.388 respectively, P < 0.05). Conclusion Through the way of stimulating PAM ,SiO 2 could inhibit GJIC of fibroblasts,and induce fibroblast proliferation.In the pathogenesis of silicotic fibrosis,the downregulation of GJIC of the pulmonary fibroblasts may play an important role.