朊病毒感染对细胞中三羧酸循环关键催化酶的影响研究

Effects of Prion Infection on Levels of the Key Catalytic Enzymes in the Tricarboxylic-acid Cycle in Cells

为了研究朊病毒感染对细胞氧化供能的主要途径—三羧酸循环关键催化酶的影响。使用ATP检测试剂盒检测朊病毒感染后细胞的ATP表达情况,采用蛋白免疫印迹方法检测朊病毒感染细胞中三羧酸循环关键催化酶的表达情况;使用细胞免疫荧光对催化酶进行细胞定位;应用线粒体分离试剂盒分离线粒体,检测线粒体中催化酶的表达情况。结果显示朊病毒感染后的细胞ATP表达水平明显降低,进一步检测三羧酸循环过程中三个主要的催化酶,发现这三个催化酶在朊病毒感染的细胞中表达量明显降低,免疫荧光实验显示催化酶与线粒体标志蛋白具有空间共定位现象,并发现在朊病毒感染细胞的线粒体中这三个催化酶的表达也显著降低。本研究发现朊病毒感染细胞中三羧酸循环催化酶的表达量明显降低,导致ATP产能下降,影响细胞代谢水平。

We measured levels of the key catalytic enzymes of the tricarboxylic acid cycle(TCAC),which is the main pathway of oxidative-energy supply in cells after prion infections. Levels of adenosine triphosphate(ATP)in prion-infected cells were measured by an ATP detection kit. Levels of the key catalytic enzymes of the TCAC in prion-infected cells were measured by western blotting. Immunofluorescence of cells was used to test the location of catalytic enzymes. A mitochondrial isolation kit was used to isolate mitochondria and measure levels of catalytic enzymes within them. ATP levels were reduced significantly in prion-infected cells. Levels of three enzymes were reduced in prion-infected cells. Catalytic enzymes were colocalized with mitochondrial proteins according to immunofluorescence analyses. Extraction of mitochondrial components from cells revealed the levels of these three catalytic enzymes to be reduced significantly in the mitochondria of prion-infected cells. This study found levels of catalyzing enzymes in the TCAC decreased significantly after prion infection,which resulted in a decrease in ATP production and affected cell metabolism.