摘要
【目的】观察荷瘤状态下小鼠心肌损伤情况,探讨park2基因编码的E3泛素-蛋白连接酶Parkin介导线粒体自噬在荷瘤小鼠心肌损伤中的作用及机制。【方法】通过PCR和蛋白质免疫印迹筛选和鉴定park2^(-/-)小鼠;通过皮下移植瘤技术构建荷瘤动物模型,采用小动物心脏超声分析小鼠心脏结构与功能,观察心室内径、心室容积、射血分数、缩短分数以及心室壁厚度的变化;采用病理学方法分析小鼠心脏的形态学特征;使用Fluo-3、Rhod-2、DHE-ROS及JC-1探针进行线粒体损伤指标检测;通过qPCR技术分析线粒体生成相关因子tfam,nrf1,线粒体融合相关因子opa1,mfn2,线粒体分裂相关因子fis1,线粒体自噬相关因子park2表达改变;通过Western Blot技术检测关键蛋白LC3Ⅱ、LC3Ⅰ、Parkin表达水平。【结果】心脏超声检测结果显示,B16黑色素瘤荷瘤小鼠心脏功能下降明显(P<0.01);电镜结果显示,荷瘤状态下小鼠心肌细胞线粒体数量增多,出现空泡化改变(P=0.032);进一步分析显示荷瘤状态可引起心肌细胞线粒体损伤,并激活线粒体自噬;park2^(-/-)小鼠荷瘤后,其死亡率明显增加(P<0.05);对park2^(-/-)小鼠进行上述荧光探针、病理学观察及关键分子检测,结果显示park2^(-/-)小鼠线粒体损伤较WT小鼠严重,线粒体自噬过程受到抑制(P<0.01)。【结论】E3泛素-蛋白连接酶Parkin在荷瘤状态心肌损伤过程中具有一定的保护作用,这与其介导的线粒体自噬有关。
【Objective】To observe the myocardial injury in tumor-bearing mice,and to explore the role and mecha⁃nism of park2 gene-encoded E3 ubiquitin protein ligase Parkin-mediated mitophagy in myocardial injury of tumor-bearing mice.【Methods】PCR and Western Blot were used to verify park2^(-/-)mice,the tumor-bearing animal models were construct⁃ed by subcutaneous transplantation,small animal ultrasound imaging system was used to monitor cardiac structure and function,the changes in left ventricular internal dimensions(LVD),left ventricular volume(LVV),ejection fraction(EF),fractional shortening(FS),and left ventricular wall thickness(LVW)were measured.Morphological changes of hearts were analyzed by pathological techniques.Fluo-3,Rhod-2,DHE-ROS and JC-1 probes were used for the detec⁃tion of mitochondrial damage indicators.qPCR technology was used to evaluate mitochondriogenesis related factors tfam and nrf1,mitochondrial fusion related factors opa1 and mfn2,mitochondrial fission related factor fis1,and mitophagy re⁃lated gene park2.Expression of LC3Ⅱ,LC3Ⅰand Parkin was assessed by Western Blot.【Results】The small animal ul⁃trasound imaging revealed that B16 melanoma-bearing mice had a significant reduction in cardiac function(P<0.01).Transmission electron microscopy(TEM)showed increased numbers of mitochondria and mitochondria vacuolization in tu⁃mor-bearing mice(P=0.023).Further molecular analysis indicated that tumor-bearing state could cause mitochondrial damage in cardiomyocytes and activate mitophagy.There was significantly increased mortality in tumor-bearing park2^(-/-)mice(P<0.05).Mitophagy was inhibited in park2^(-/-)mice and more severe mitochondrial damage was noted(P<0.05).【Conclusion】E3 ubiquitin protein ligase Parkin plays a protective role in cardiomyocyte injury at a tumor-bearing state,which is associated with E3 ubiquitin protein ligase Parkin-mediated mitophagy.
作者
邓世杰
李兴会
蔡卫斌
DENG Shi-jie;LI Xing-hui;CAI Wei-bin(Guangdong Provincial Engineering and Technology Research Center for Disease-Model Animals//Laboratory Animal Center,Sun Yat-sen University,Guangzhou 510006,China;Department of Biochemistry and Molecular Biology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China)
出处
《中山大学学报(医学科学版)》
CAS
CSCD
北大核心
2022年第2期181-191,共11页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家重点研发计划(2019YFA0801403)
国家自然科学基金(82170261,81970219,82000250)
广东省自然科学科学基金(2021A1515011005,2021B1212040006)
中国博士后基金(2020M672976)。