缺氧条件下GC-1细胞凋亡的作用机制研究

Study on Mechanism of GC-1 Apoptosis Under Hypoxic Conditions

以小鼠精原细胞系GC-1细胞为研究对象,探讨缺氧条件下GC-1细胞凋亡潜在的分子机制。首先,采用CCK-8(Cell Counting Kit-8)法检测不同缺氧时间处理下的细胞活力,以确定细胞缺氧损伤的时间;然后,通过化学荧光法检测GC-1细胞中活性氧(reactive oxygen species,ROS)的含量,采用JC-1法检测线粒体的膜电位,采用比色法检测ATP的含量,采用线粒体荧光探针法检测线粒体的数量与分布,并采用透射电镜观察细胞的超微结构;最后,利用实时荧光定量PCR检测线粒体信号通路相关基因胱天蛋白酶(caspase)-3、caspase-9、细胞色素c(cytochrome c,Cyt-c)、Bax和Bcl-2的mRNA表达水平。结果显示,缺氧36 h后,细胞活力为(60.36±5.40)%,符合后续实验需求;在缺氧条件下,GC-1细胞中的ROS含量显著增加,ATP含量显著下降,线粒体膜电位下降、数量减少,同时细胞中促凋亡相关基因的表达水平上调,抗凋亡因子Bcl-2的基因表达水平下调。实验结果初步表明,缺氧可导致GC-1细胞线粒体功能障碍,ROS/线粒体信号通路是缺氧导致GC-1细胞凋亡可能的分子机制。

The potential molecular mechanism of apoptosis of mouse spermatogonial GC-1 cells under hypoxia was investigated.First,the cell viability under hypoxia for different time periods was measured using the Cell Counting Kit-8(CCK-8)to determine the time of cell damage induced by hypoxia.Then,the content of reactive oxygen species(ROS)in GC-1 cells was detected by chemical fluorescence method,the membrane potential of mitochondria was detected by JC-1 assay,and the content of ATP was detected by colorimetry.Meanwhile,the number and distribution of mitochondria were detected using fluorescent probe method,and the ultrastructure of the cells was observed by transmission electron microscopy.Finally,mRNA expression levels of mitochondrial signaling pathway related genes caspase-3,caspase-9,cytochrome c(Cyt-c),Bax and Bcl-2 were analysed using real-time fluorescence quantitative PCR.The results showed that,after 36 h of hypoxia,the cell viability was(60.36±5.40)%,which met the requirement of subsequent experiments.Under hypoxia conditions,the ROS content in GC-1 cells increased significantly,and the ATP content decreased significantly.The membrane potential and number of mitochondria were reduced.At the same time,the expression levels of pro-apoptosis-related genes were up-regulated,and the gene expression level of antiapoptotic factor Bcl-2 was down-regulated.The results showed that hypoxia can lead to mitochondrial dysfunction in GC-1 cells,and ROS/mitochondrial signaling pathway may be the molecular mechanism of hypoxia-induced apoptosis of GC-1 cells.