Blau综合征细胞模型的建立

Establishment of cell models for Blau syndrome

目的建立稳定可靠的Blau综合征(BS)体外细胞模型。方法以小鼠腹腔巨噬细胞系(RAW264.7)、原代骨髓巨噬细胞(iBMDM)和人急性单核细胞白血病细胞系(THP-1)为研究对象,用胞壁酰二肽(MDP)或MDP衍生物(L18-MDP)刺激细胞建立模型,同时设置TNF-α抑制剂依那西普(ETN)和R1P2抑制剂(GSK583)处理组考察模型对药物的响应以评价其有效性,22 h后收集细胞培养上清,ELISA检测细胞上清中肿瘤坏死因子α(TNF-α)水平。结果经10μg/mL MDP刺激后,RAW264.7和iBMDM细胞上清中TNF-α含量显著升高(P<0.01),ETN和GSK583可降低MDP诱导的TNF-α水平升高(P<0.01)。THP-1细胞经PMA诱导为THP-1-Mφ细胞株后,0.2和1μg/mL L18-MDP均可增加上清中TNF-α的水平(P<0.01);在给予0.2μg/mL L18-MDP刺激的同时加入ETN可显著抑制TNF-α的分泌水平(P<0.001)。结论本实验用RAW264.7、iBMDM和THP-1细胞建立了良好的BS体外细胞模型,模型具有简单方便、容易重复操作以及可控性强的优点,这为进一步研究BS的发病机制以及筛选和评估治疗药物奠定基础。

Objective To establish a stable and reliable cell models of Blau syndrome(BS)in vitro.MethodsRAW264.7,iBMDM and THP-1 cells were used as the research objects and then stimulated by muramyl dipeptide(MDP)or L18-MDP.Meanwhile,positive drugs etanercept(ETN)and GSK583 treatment groups were set to investigate the response of the model to evaluate effectiveness.Cell culture supernatant was collected after 22 h,and the level of tumor necrosis factor-α(TNF-α)was determined by enzyme linked immunosorbent assay(ELISA).Results The secretion of TNF-αfrom RAW264.7 and iBMDM cells was significantly increased after stimulation by 10μg/mL MDP(P<0.01),while ETN and GSK583 inhibited the increase of TNF-αinduced by MDP(P<0.01).After THP-1 cells were induced into THP-1-Mφby PMA,0.2 and 1μg/mL L18-MDP increased the level of TNF-αin the culture supernatant(P<0.01).When ETN was added along with 0.2μg/mL L18-MDP,the secretion of TNF-αwas significantly inhibited(P<0.01).Conclusions RAW264.7,iBMDM and THP-1 cells may develop good cell models for BS in vitro.The model has the advantages of simplicity,convenience,easy repetition and strongcontrollability,which lay a foundation for further research on the pathogenesis of BS and screening and evaluation of therapeutic drugs.