烟雾提取物对转化生长因子β_(1)刺激的人胚肺成纤维细胞增殖及分化的影响

Effects of smoke extract on the proliferation and differentiation of human embryonic lung fibroblasts induced by transforming growth factor-β_(1)

目的研究烟雾提取物(SE)对转化生长因子β_(1)(TGF-β_(1))刺激的人胚肺成纤维细胞MRC-5增殖及分泌羟脯氨酸(HYP)、Ⅰ型胶原蛋白(ColⅠ)、Col Ⅲ及α平滑肌肌动蛋白(α-SMA)的影响。方法本研究为实验研究。采用完全培养基将通过自制装置提取出的烟雾成分制备成不同浓度(0%、1%、2%、5%、10%)的SE溶液, 选用MRC-5细胞进行细胞培养, 将其分为对照组和TGF-β_(1)组。TGF-β_(1)组在TGF-β_(1)刺激MRC-5细胞1 d后用含不同浓度SE的培养基继续培养48 h, 对照组采用普通培养基和不同浓度SE的培养基培养。通过CCK-8试剂检测细胞活力, 观察不同浓度SE对细胞数量、大小、形态及胶原合成的影响, 并制作细胞爬片, 天狼星红染色后行胶原定量检测, 最后ELISA法检测HYP、Col Ⅰ、Col Ⅲ、α-SMA水平, qRT-PCR检测Col Ⅰ、Col Ⅲ、α-SMA mRNA表达水平。结果对照组中, 不同浓度SE均抑制细胞的增殖, 抑制胶原的合成, 抑制HYP、Col Ⅰ、Col Ⅲ、α-SMA的水平, 且随着浓度增加, 抑制作用越明显;TGF-β_(1)组中, 1%、2%的SE促进细胞的增殖, HYP、ColⅠ、Col Ⅲ、α-SMA的水平增加, 且2%的SE最显著, 5%的SE对细胞的影响不显著, 但10%的SE抑制细胞的增殖, 抑制胶原的表达, Col Ⅰ、Col Ⅲ、α-SMA的水平下降。结论低浓度SE通过TGF-β_(1)介质促进MRC-5细胞增殖与分化, 高浓度SE对MRC-5细胞的增殖与分化起抑制作用, 提示TGF-β_(1)是烟雾吸入损伤后肺纤维化的重要中间介质。

Objective To explore the effects of smoke extract(SE)on the proliferation of human embryonic lung fibroblasts MRC-5 and on the secretion of hydroxyproline(HYP),collagen typeⅠ(ColⅠ),ColⅢ,andα-smooth muscle actin(α-SMA)induced by transforming growth factorβ_(1)(TGF-β_(1)).Methods This was an experimental study.The complete medium was used to prepare SE solutions with different concentrations(0%,1%,2%,5%,and 10%)of the smoke components extracted through a homemade device,and MRC-5 cells were selected for cell culture,which were divided into control and TGF-β_(1) groups.The TGF-β_(1) group was cultured with medium containing different concentrations of SE for 48 h after 1 d of TGF-β_(1) stimulating MRC-5 cells,and the control group was cultured with normal medium and medium with different concentrations of SE.Then CCK-8 reagent was used to detect the cell viability of MRC-5 cells.The effects of different concentrations of SE on cell count,size,morphology,and collagen synthesis were also observed.After producing cell slides,sirius red staining was used for collagen quantitative detection,ELISA method was used to detect the levels of HYP,ColⅠ,ColⅢ,andα-SMA,and qRT-PCR was used to detect the mRNA levels of ColⅠ,ColⅢ,andα-SMA.Results In the control group,the cell proliferation,collagen synthesis,and the levels of HYP,ColⅠ,ColⅢ,andα-SMA were all inhibited by different concentrations of SE.As the concentration of SE increased,the inhibitory effect was more obvious.In the TGF-β_(1) group,1%and 2%SE promoted cell proliferation and increased the levels of HYP,ColⅠ,ColⅢ,andα-SMA,with the effect of 2%SE more significant.5%SE had no significant effect on cells,but 10%SE inhibited the proliferation of MRC-5 cells and the expression of collagen,and decreased the levels of ColⅠ,ColⅢ,andα-SMA.Conclusions Low concentrations of SE promote the proliferation and differentiation of MRC-5 cells through TGF-β_(1) mediators,while high concentrations of SE inhibit the proliferation and differentiation of MRC-5 cells,suggesting that TGF-β_(1) is an important intermediate mediator of lung fibrosis after smoke inhalation injury.