摘要
目的基于NOTCH信号通路探讨FIZZ1对人胚肺成纤维细胞(HELF)转分化为肌成纤维细胞(MFb)的影响。方法1)HELF培养与传代后分成5组,加入不同浓度的FIZZ1(0.25ug/mL,0.5 ug/mL,1ug/mL,2ug/mL),采用CCK8实验检测细胞增殖活性。2)用不同浓度DAPT(0.25umol/L,0.5umol/L)处理4小时,再加入1ug/mL FIZZ1处理24小时,用CCK8法检测细胞增殖活性确定DAPT的最佳实验浓度。3)HELF分成3组:对照组(无血清培养基4h+2%PBS处理24h)、模型组(无血清培养基4h+1ug/mLFIZZ1处理24h)、DAPT组(含0.5umol/L DAPT的无血清培养基4h+1ug/mL FIZZ1处理24h),RT-PCR检测HELF中a-SMA、NOTCH的mRNA表达。结果1)FIZZ1刺激HELF后,与空白组相比,细胞核质比缩小,变成长梭形,数量增多。2)CKK8实验结果显示1ug/mL FIZZ1组增值率(OD值)最高(P<0.05﹚。用0.5umol/L浓度DAPT处理后细胞增殖率下降最为显著。3)RT-PCR实验显示,用1ug/mLFIZZ1刺激HELF后,与对照组相比,a-SMA及NOTCH1的mRNA表达显著增高(P<0.05﹚,0.5umol/L浓度DAPT处理后其两者表达显著降低。结论FIZZ1可能通过NOTCH信号通路的激活促进成纤维细胞向肌成纤维细胞的转化增殖,导致a-SMA分泌增多,加重肺间质纤维化的发生发展。
Objective To investigate the effect of FIZZ1 on human embryonic lung fibroblasts(HELF)transforming into myofibroblasts(MFB)based on Notch signaling pathway.Methods 1.After HELF culture and subculture,the cells were divided into five groups.Different concentrations of FIZZ1(0.25ug/mL,0.5ug/mL,1ug/mL,2ug/mL)were added.The cell proliferation activity was detected by CCK8.2.DAPT with different concentrations(0.25umol/l,0.5umol/L)was treated for 4 hours,and then added with 1ug/mL FIZZ1 for 24 hours.The best experimental concentration of DAPT was determined by CCK8 method.3.HELF was divided into three groups:the control group(serum-free medium 4H+2%PBS treatment 24h),the model group(serum-free medium 4H+1ug/mL FIZZ1 treatment 24h),and the DAPT group(serum-free medium 4H+1ug/mL FIZZ1 treatment 24h with 0.5umol/L DAPT).RT-PCR was used to detect the mRNA expression of a-SMA and Notch in HELF.Results 1.Compared with the control group,the ratio of cell nucleus to cytoplasm decreased and became long fusiform,and the number increased.Ckk8 experimental results showed that the highest value-added rate(ODV)was found in the 1ug/mL FIZZ1 group(P<0.05).DAPT with a concentration of 0.5 umol/L decreased the cell proliferation rate most significantly.3.RT-PCR showed that the mRNA expression of a-SMA and Notch1 in HELF was significantly higher than that in the control group(P<0.05).Conclusion FIZZ1 may promote the transformation and proliferation of fibroblasts to myofibroblasts through the activation of Notch signaling pathway,which may increase the secretion of a-SMA and aggravate the occurrence and development of pulmonary interstitial fibrosis.
作者
徐芳
王爱利
黄莺
XU Fang;WANG Ai-li;HUANG Ying(Department of Respiratory Diseases,Wuhan No.1 Hospital,Wuhan,Hubei430022,China)
出处
《临床肺科杂志》
2020年第12期1885-1888,共4页
Journal of Clinical Pulmonary Medicine
基金
湖北省武汉市卫计委专项基金资助项目(No.WZ14D09)。
