OXSR1活性对p53依赖和非依赖途径介导的犬肾细胞凋亡的影响  

Effects of OXSR1 Activity on Apoptosis of Madin-Darby Canine Kidney Cells Through p53-dependent and-independent Pathways

作　　者：曾红玉 叶贵珊 武琦 张安定[1] 韩丽[1] ZENG Hongyu;YE Guishan;WU Qi;ZHANG Anding;HAN Li(College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学动物医学院,武汉430070

出　　处：《中国畜牧兽医》2024年第10期4222-4234,共13页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(32272974)。

摘　　要：【目的】研究氧化应激反应激酶1(oxidative stress-responsive 1,OXSR1)在犬肾细胞(Madin-Darby canine kidney cell,MDCK)凋亡中的作用,为深入理解MDCK凋亡通路及研究以OXSR1基因为靶向的MDCK凋亡的干预疗法提供参考。【方法】利用CRISPR/Cas9技术在MDCK-Cas9细胞上构建OXSR1基因敲除细胞系,在OXSR1基因敲除细胞中转入OXSR1基因回补与失活质粒以构建OXSR1基因回补与失活细胞系。以刺激剂MMC、JNJ-26854165、Rotenone分别刺激野生型细胞与OXSR1基因敲除、回补、失活细胞,通过流式细胞术检测激活的p53依赖和非依赖途径、ROS信号通路介导的细胞凋亡,并通过实时荧光定量PCR检测相关凋亡基因的表达情况。【结果】靶序列出现明显的Indel特征,证实OXSR1基因敲除细胞系构建成功。Western blotting检测显示,OXSR1基因回补与失活细胞系构建成功。流式细胞术发现,刺激剂MMC、JNJ-26854165、Rotenone均能诱导细胞发生不同程度的凋亡,其中在MMC和Rotenone刺激下,OXSR1基因敲除细胞凋亡率极显著或显著低于野生型细胞和OXSR1基因回补细胞(P<0.01;P<0.05),与OXSR1基因失活细胞无显著差别(P>0.05);在JNJ-26854165刺激下,OXSR1基因敲除细胞凋亡率极显著或显著低于野生型细胞、OXSR1基因回补和失活细胞(P<0.01;P<0.05)。实时荧光定量PCR检测发现,当MMC和Rotenone刺激细胞时,OXSR1基因敲除细胞的BCL-XL基因表达量极显著或显著高于野生型细胞和OXSR1基因回补细胞(P<0.01;P<0.05),与OXSR1基因失活细胞无明显差别(P>0.05);当JNJ-26854165刺激细胞时,OXSR1基因敲除细胞的BCL-XL基因表达量极显著高于野生型细胞、OXSR1基因回补与失活细胞(P<0.01),NOXA基因表达量极显著低于野生型细胞、OXSR1基因回补与失活细胞(P<0.01)。【结论】OXSR1基因及其激酶活性可调节MMC、JNJ-26854165、Rotenone刺激MDCK时BCL-XL基因转录,影响p53依赖和非依赖途径以及ROS途径介导的细胞凋亡。【Objective】This study was aimed to investigate the role of oxidative stress-responsive 1(OXSR1)in the apoptosis of Madin-Darby canine kidney cells(MDCK),so as to provide a reference for better understanding the apoptotic pathway in MDCK and study on interventional therapies targeting OXSR1 gene for MDCK apoptosis.【Method】OXSR 1 gene knockout cell lines were produced using CRISPR/Cas9 technology on MDCK-Cas9 cells.OXSR 1 gene competent and inactivae cell lines were generated by transfecting OXSR 1 gene backtill and inactivation plasmids into OXSR 1 gene knockout cells.MMC,JNJ-26854165 and Rotenone were used to stimulate wild-type,OXSR 1 gene knockout,gene competent and inactive cells,and the apoptosis through p53-dependent and-independent pathways and ROS signaling pathway were detected using flow cytometry.The expression of relevant apoptotic genes were detected by Real-time quantitative PCR.【Result】There was apparent Indel features in the target sequences,it confirmed OXSR 1 gene knockout cell lines were successfully constructed.Western blotting results indicated that OXSR 1 gene competent and inactive cell lines were successfully constructed.Flow cytometry determined that MMC,JNJ-26854165 and Rotenone could induce varying degrees of cell apoptosis.When stimulated with MMC and Rotenone,the apoptosis rate of OXSR 1 gene knockout cells was extremely significantly or significantly lower than that of wild-type and OXSR 1 gene competent cells(P<0.01 or P<0.05),and there was no significant difference with OXSR 1 gene inactive cells(P>0.05).When stimulated with JNJ-26854165,the apoptosis rate of OXSR 1 gene knockout cells was extremely significantly or significantly lower than that of wild-type,OXSR 1 gene competent and inactive cells(P<0.01 or P<0.05).Real-time quantitative PCR results showed that when cells were stimulated with MMC and Rotenone,the expression of BCL-XL gene in OXSR 1 gene knockout cells was extremely significantly or significantly higher than that of wild-type and OXSR 1 gene competent ce

关 键 词：OXSR1基因 p53依赖和非依赖途径 ROS信号通路 MDCK 细胞凋亡 

分 类 号：S852.23[农业科学—基础兽医学]