博来霉素和内毒素对鼠肺成纤维细胞增殖及表型转化的影响

Effect of bleomycin and lipopolysaccharide on proliferation and fibroblast-to-myofibroblast differentiation of rat lung fibroblasts

目的探讨博来霉素(BLM)和内毒素(LPS)对肺成纤维细胞增殖及表型转化的影响及可能机制。方法分离、鉴定新生SD大鼠肺成纤维细胞,分为对照组、BLM组和LPS组。实验组加入不同浓度梯度的BLM(0.0001、0.001、0.01、0.1、1 mg/L)和LPS(0.01、0.1、1、10、100 mg/L)。分别采用四甲基偶氮唑蓝(MTT)法检测细胞增殖,酶联免疫吸附试验检测细胞上清液中转化生长因子β1(TGF-β1)蛋白含量。根据MTT结果进一步将BLM/LPS组分为低浓度组(分别为0.001、0.1 mg/L)和高浓度组(分别为1、10 mg/L),并且采用蛋白质印迹法检测α平滑肌肌动蛋白(α-SMA)表达,透射电镜观察细胞超微结构,单细胞凝胶电泳检测DNA损伤。结果HE染色显示原代培养的细胞呈成纤维细胞典型的纺锤形,免疫组织化学法鉴定示波形蛋白表达阳性。与对照组相比,在一定浓度范围内,BLM组和LPS组细胞增殖增高(P<0.05),且前者更显著;在较低浓度时,BLM组TGF-β1含量明显增高(P<0.05),随着浓度的增加,TGF-β1含量减少(P<0.05);而LPS各组TGF-β1含量比较差异无统计学意义。BLM/LPS低浓度组α-SMA蛋白表达增多(P<0.05);高浓度组α-SMA蛋白表达均明显减少(P<0.05),BLM组更显著。透射电镜显示,BLM低浓度组可见细胞核呈分叶状;BLM高浓度组细胞器明显水肿;LPS低浓度组和高浓度组均可见水肿的细胞器及分叶状细胞核。单细胞凝胶电泳结果显示,BLM与LPS低浓度组均可见明显彗星尾,二者彗星尾长差异无统计学意义;高浓度组彗星尾长进一步增加,且BLM组更明显。结论在一定浓度范围内,BLM和LPS均可促进肺成纤维细胞增殖及表型转化,且前者作用明显强于后者;BLM和LPS均可致不同程度的肺成纤维细胞DNA损伤,这可能与肺成纤维细胞不同程度增殖及表型转化有关。

Objective To investigate the effect of bleomycin(BLM)and lipopolysaccharide(LPS)on the proliferation and fibroblast-to-myofibroblast differentiation of rat lung fibroblasts and the possible mechanism.Methods Primary lung fibroblasts were isolated and purified from new born Sprague Dawley rats.Fibroblasts were divided into control group,BLM-treated group and LPS-treated group.In the experimental groups,the fibroblasts were exposed to the concentration of 0.0001,0.001,0.01,0.1,1 mg/L BLM and 0.01,0.1,1,10,100 mg/L of LPS respectively.The proliferation of fibroblasts and the secretion of transforming growth factor-β1(TGF-β1)were evaluated by methyl thiazolyl tetrazolium(MTT)assay and enzyme linked immunosorbent assay,respectively.The concentration of 0.001/0.1 mg/L and 1/10 mg/L were further defined as low-dose and high-dose in BLM/LPS group according to the MTT assay.The expression ofα-smooth muscle actin(α-SMA),ultrastructural morphology and DNA damage of fibroblasts were assessed by Western blot,transmission electron microscopy and single cell gel electrophoresis in the above groups.Results HE staining showed primary cultured cells had typical morphology of fibroblast with a spindle shape.Immunohistochemical staining showed the expression of vimentin was positive.Compared with control group,the proliferation of fibroblasts was elevated at certain concentrations in BLM and LPS groups(P<0.05),this effect was significantly greater in BLM-treated group.The level of TGF-β1 was increased in low-dose of BLM group(P<0.05),which was decreased as the concentration became higher(P<0.05).However,the level of TGF-β1 was unchanged in different concentrations of LPS-treated groups.The expression ofα-SMA was significantly increased in the low-dose of BLM/LPS group(P<0.05),but markedly reduced in the high-dose of BLM/LPS group(P<0.05),which was more obvious in BLM-treated group.Transmission electron microscopy showed that the nucleus was shapeless in the low-dose of BLM group,the organelles were evidently dilated in the high-dose of BLM group.However,the dilated organelles and shapeless nucleus were noted in low/high-dose of LPS group.Single cell gel electrophoresis showed that the length of the tail was significantly increased in the low-dose of BLM/LPS group and there was no significant difference between them.However,high-dose of BLM/LPS group displayed a more accentuated comet tail,especially in BLM-treated group.Conclusions BLM and LPS can promote the proliferation of fibroblasts and fibroblast-to-myofibroblast differentiation at a certain concentration,and the former has more obvious effect than the latter.BLM and LPS can both induce DNA damage of fibroblasts to a different extent,which may be related to the proliferation and differentiation of fibroblasts.