非经典细胞焦亡体外模型的构建

Reconstruction of Noncanonical Pyroptosis in vitro

旨在深入研究非经典细胞焦亡的激活机制,本研究在体外构建非经典细胞焦亡模型,利用PCR方法扩增人源目的基因Caspase-4、hGSDMD-FL和hGSDMD-p30,使用无缝克隆的方法连接到相应的载体中,构建重组真核表达质粒pCMV-MYC-Caspase-4、p3XFLAG-hGSDMD-FL和pcDNA3.1-hGSDMD-p30-MYC,利用VigoFect试剂转染到HEK293T细胞后,采用Western blot方法验证各蛋白的表达情况;通过质粒共转染的方法构建非经典的细胞焦亡模型,分别检测细胞上清中LDH的释放、GSDMD有无被切割为有活性的N端p30片段和荧光观察PI染色情况来确定非经典细胞焦亡模型是否构建成功。结果显示:pCMV-MYC-Caspase-4、p3XFLAG-hGSDMD-FL重组质粒均成功构建,各蛋白于HEK293T细胞内均成功表达,质粒共转染后LDH分泌显著升高,Caspase-4将GSDMD全长片段切割为有活性N端GSDMD-p30片段,荧光显微镜观察到明显的PI染色,表明非经典细胞焦亡模型体外构建成功。非经典细胞焦亡模型在体外的成功构建,为进一步研究其激活机制奠定了基础。

To reconstruct noncanonical pyroptosis in vitro,the coding sequences of Caspase-4,hGSDMD-FL and hGSDMD-p30 were amplified by PCR,and these PCR products were inserted into corresponding plasmids to get the following recombinant eukaryotic expression vectors:pCMV-MYC-Caspase-4,p3XFLAG-hGSDMD-FL.The expressions of both recombinant vectors in HEK293T cells were confirmed by Western blot.Whether the noncanonical pyroptosis model was successfully constructed was confirmed by testing the production of LDH,active N-terminal GSDMD-p30 and PI staining after transfection into HEK293T cells.The results showed that recombinant vectors including pCMV-MYC-Caspase-4,p3XFLAG-hGSDMD-FL,and pcDNA3.1-hGSDMD-p30-MYC were constructed and each protein was successfully expressed in HEK293T cells.After co-transfection,significant production of LDH was detected,Caspase-4 cleaved the full-length GSDMD into active N-terminal GSDMD-p30 domain and obvious PI staining was observed by fluorescence microscope,which indicated that the noncanonical pyroptosis was successfully reconstructed in vitro.Therefore,the noncanonical pyroptosis is reconstructed in vitro,which establishes foundation for further revealing the mechanism of noncanonical pyroptosis activation.