硫化氢对人胚肺成纤维细胞缺氧后增殖活力及细胞凋亡的影响

Effects of hydrogen sulphide on the proliferation and apoptosis of human fetal lung fibroblasts during hypoxia

目的探讨内源性及外源性硫化氢(H2S)对缺氧诱导下人胚肺成纤维细胞增殖效应及凋亡率的影响。方法以2%O2-93%N2-5%CO2体外培养人胚肺成纤维细胞24h,制备细胞缺氧模型。细胞培养分为6组:①缺氧(N2)组;②N2+600μmol.L-1NaHS组;③N2+1200μmol.L-1NaHS组;④N2+6400μmol.L-1NaHS组;⑤N2+400μmol.L-1L-半胱氨酸(Cys)组;⑥N2+200μmol.L-1S-腺苷甲硫氨酸(SAM)组。各组细胞24h缺氧培养后,采用MTT法和流式细胞术分别检测细胞增殖活力和细胞凋亡率。结果与N2组相比,600和1200μmol.L-1NaHS(H2S供体)可降低缺氧后人胚肺成纤维细胞的增殖活力(P<0.01),但对细胞凋亡率无影响(P>0.05);而6400μmol.L-1NaHS虽然不影响细胞缺氧后的增殖活力(P>0.05),但却增加其凋亡率(P<0.05);胱硫醚-β-合酶(H2S合成酶)底物Cys和激动剂SAM对缺氧后人胚肺成纤维细胞增殖活力无影响(P>0.05);但它们却使缺氧后人胚肺成纤维细胞凋亡率高于N2组(P<0.05)。结论内源性及外源性H2S可降低缺氧诱导的人胚肺成纤维细胞增殖活力、促进细胞凋亡,提示内源性H2S还可能通过肺成纤维细胞,抑制缺氧导致的肺血管结构重建发挥其保护作用。

Aim To investigate the effects of endogenous and exogenous hydrogen sulfide（H2S）on the proliferation and apoptosis of human fetal lung fibroblasts.Methods Human fetal lung fibroblasts were cultured under 2% O2~93% N2~5% CO2 for 24 h to produce hypoxia.Cells were divided into 6 groups：（1）Hypoxia group（N2）;（2）N2＋600 μmol·L-1 NaHS group;（3）N2＋1 200 μmol·L-1 NaHS group;（4）N2＋6 400 μmol·L-1 NaHS group;（5）N2＋400 μmol·L-1 cysteine（Cys）group;（6）N2＋200 μmol·L-1 S-adenosyl-L-methionine（SAM）group.After they were cultured for 24 h,MTT assay was used to evaluate the cell proliferation,and flow cytometry was used to detect cell apoptosis.Results Compared with N2 group,600 and 1 200 μmol·L-1 NaHS（H2S donor）significantly reduced proliferation induced by hypoxia of human fetal lung fibroblasts（P0.01）without effects on apoptotic rates of cells（P0.05）and 6 400 μmol·L-1 NaHS increased apoptosis of human fetal lung fibroblasts during hypoxia significantly（P0.05）,although no effects were found on proliferation of cells（P0.05）.In addition,Cys,substrate of cystathionine β-synthetase（H2S synthase,CBS）or SAM（activator of CBS）did not affect proliferation of human fetal lung fibroblasts induced by hypoxia（P0.05）,whereas apoptotic rates were increased significantly compared with that of N2 group（P0.05）.Conclusions Endogenous and exogenous hydrogen sulfide can inhibit proliferation induced by hypoxia and promote apoptosis of human fetal lung fibroblasts,suggesting endogenous hydrogen sulfide may play a protective role through lung fibroblasts by inhibiting the pulmonary vascular structural remodeling caused by hypoxia.