经典途径细胞焦亡模型的构建

Construction ofcanonical pyroptosis model

细胞焦亡是一种具有促炎性的细胞程序性死亡过程,可分为依赖Caspase-1的经典途径和依赖Caspase-4/5/11的非经典途径,广泛参与肿瘤、感染性疾病、代谢性疾病等疾病的发生与发展。为构建经典途径细胞焦亡的体外模型,通过PCR扩增目的基因Pro-Caspase-1、H-GSDMD-FL和H-GSDMD-P30,分别连接到相应的载体中,构建重组真核表达质粒p3×FLAG-CMV-Caspase-1、Myc-CMV-hGSDMD-FL和pcDNA3.1-Myc-hGSDMD-p30。利用Lipo8000^(TM)试剂将重组质粒转染到HEK293T细胞中,通过Western blot方法验证蛋白表达情况;进一步将质粒共转染至HEK293T细胞24 h后,检测上清中LDH释放情况,提取蛋白后通过Western blot方法验证蛋白表达及切割情况,并且进行PI染色观察细胞膜破坏情况,验证经典途径的细胞焦亡体外模型是否成功构建。结果显示,重组真核表达质粒均成功构建,p3×FLAG-CMV-Caspase-1和Myc-CMV-hGSDMD-FL可在HEK293T细胞中成功表达。质粒共转染后Western blot检测到GSDMD-FL被Caspase-1切割,切割条带与GSDMD-p30大小相符,上清中LDH水平明显升高,与GSDMD-p30单独转染结果相似,PI染色结果也与上述情况一致,表明HEK293T细胞中经典途径细胞焦亡模型的成功构建,为进一步探究焦亡激活机制以及开发相关药物提供了体外模型。

Pyroptosis is a proinflammatory form of programmed cell death,which can be divided into canonical pyroptosis and noncanonical pyroptosis,mediated by Caspase-1 or Caspase-4/5/11.It is widely involved in the occurrence and development of cancer,infectious diseases,metabolic diseases and so on.In order to construct the canonical pyroptosis model in vitro,the coding sequences of pro-Caspase-1,H-GSDMD-FL and H-GSDMD-p30 were amplified by PCR and ligated into corresponding vectors respectively.The recombinant eukaryotic expression plasmids p3×FLAG-CMV-Caspase-1,Myc-CMV-hGSDMD-FL and pcDNA3.1-Myc-hGSDMD-p30 were constructed and transfected into HEK293 T cells by Lipo8000^(TM) reagent.The expression of each protein was verified by Western blot.After the plasmids were cotransfected into HEK293 T cells for 24 h,the release of LDH in the supernatant was detected,the protein expression and cleavage were verified by Western blot,and the morphological changes of cell nucleus were observed by PI staining to verify whether the canonical pyroptosis model in vitro was successfully constructed.The results showed that the recombinant eukaryotic expression plasmids were successfully constructed,p3×FLAG-CMV-Caspase-1 and Myc-CMV-hGSDMD-FL were successfully expressed in HEK293 T cells.After plasmid cotransfection,Western blot detected that GSDMD-FL was cleaved by Caspase-1,the cleavage band was consistent with the size of GSDMD-p30,the LDH level in the supernatant was significantly increased,which was similar to the result of transfection of GSDMD-p30,and the PI staining result was also consistent with the above situation.The successful construction of canonical pyroptosis model in HEK293 Tcells provides an in vitro model for further exploring the mechanism of pyroptosis activation and developing related drugs.