Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (11): 1750-1757.doi: 10.12307/2023.125
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Wu Xiaolei, Han Yu, Li Jialei, Wang Shuang, Cao Jimin, Sun Teng
Received:
2022-02-10
Accepted:
2022-04-24
Online:
2023-04-18
Published:
2022-09-27
Contact:
Sun Teng, MD, Associate professor, Master’s supervisor, Key Laboratory of Cell Physiology of Ministry of Education, Shanxi Provincial Key Laboratory of Cell Physiology, Department of Physiology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
Cao Jimin, MD, Professor, Doctoral supervisor, Key Laboratory of Cell Physiology of Ministry of Education, Shanxi Provincial Key Laboratory of Cell Physiology, Department of Physiology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
About author:
Wu Xiaolei, Master candidate, Key Laboratory of Cell Physiology of Ministry of Education, Shanxi Provincial Key Laboratory of Cell Physiology, Department of Physiology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
Supported by:
CLC Number:
Wu Xiaolei, Han Yu, Li Jialei, Wang Shuang, Cao Jimin, Sun Teng. piRNA-5938 can regulate cardiomyocyte apoptosis and mitochondrial fission[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(11): 1750-1757.
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2.1 实验动物数量分析 ①小鼠模型:实验选用小鼠45只,分别作为假手术组和缺血再灌注组,其中缺血再灌注组造模成功的小鼠有21只,成功率为70%。②大鼠模型:实验选用大鼠15只,分别作为假手术组和缺血再灌注组,其中缺血再灌注组造模成功的小鼠有8只,成功率为80%。 2.2 深度测序鉴定心肌缺血再灌注模型中大量piRNA表达水平上调 缺血再灌注模型通常用于临床再灌注损伤的研究。在C57小鼠心脏中构建了缺血再灌注模型及假手术模型,进行RNA深度测序。测序结果显示,小鼠心脏中约表达1 200种piRNA,其中高表达的piRNA有10%左右。筛选出正常和缺血再灌注损伤小鼠心脏中差异表达的422种piRNA,其中约有289条piRNA在缺血再灌注模型中表达水平显著上调,133条piRNA表达水平显著下调。选取了其中18条表达水平显著上调的piRNA,对其丰度进行统计分析,见图1。经过筛选,最终以表达水平显著上调的AB350089.1(piRNA-5938)作为研究对象。"
2.4 双氧水诱导心肌细胞凋亡模型中piRNA-5938表达水平显著上调 缺血再灌注损伤发生后,损伤部位会有活性氧自由基释放,引起细胞凋亡。为模拟再灌注损伤中氧化自由基引起的细胞凋亡,使用双氧水诱导SD大鼠乳鼠原代心肌细胞凋亡,实时荧光定量PCR实验结果表明,相较于空白对照组,100 μmol/L双氧水处理后,piRNA-5938的表达水平无显著变化。加大双氧水浓度后,200 μmol/L双氧水处理组中,piRNA的表达水平上调约1.8倍(P < 0.05),500 μmol/L双氧水处理组中,piRNA-5938上调超过4倍(P < 0.01),表明原代心肌细胞中piRNA-5938的上调程度呈与双氧水浓度呈正相关,即具有浓度依赖性,见图3。"
2.7 piRNA-5938调控心肌细胞凋亡 文献报道双氧水可诱导细胞凋亡,TUNEL实验结果也证实,200 μmol/L双氧水处理组后,细胞凋亡率相对空白对照组显著提高,即低浓度的双氧水诱导了原代心肌细胞凋亡,见图6。之前的结果显示,双氧水处理心肌细胞后piRNA-5938的表达水平显著上调,因此猜测piRNA-5938可能调控了双氧水诱导的心肌细胞凋亡。TUNEL法检测心肌细胞凋亡结果显示,与阴性对照组相比,转染piRNA-5938拮抗剂的心肌细胞再经双氧水处理,发生凋亡的细胞数目显著减少,凋亡率由约34%降低到约22%,降低了30%左右(P < 0.05)。以上结果表明,抑制内源性的piRNA-5938显著抑制了双氧水诱导的心肌细胞凋亡,起到心肌保护作用。"
2.9 生物信息学方法预测piRNA-5938靶向线粒体网络调控分子miR-324和miR-668 进一步寻找piRNA-5938调控线粒体分裂的下游靶标。RNAhybrid软件分析了piRNA和microRNA序列,结果显示,piRNA-5938分别与miR-324和miR-668序列能够很好地互补配对,提示piRNA-5938在体内可能与miR-324和miR-668相互结合。已有研究表明miR-324和miR-668是线粒体动态网络调控的重要分子,miR-324通过抑制线粒体分裂调控因子Mtfr1进而抑制了线粒体分裂、心肌细胞凋亡和心肌梗死。miR-668通过靶向线粒体分裂蛋白Mtp18进而抑制了缺氧和缺血引起的线粒体分裂。因此,大胆推测piRNA-5938可能通过靶向结合miR-324或miR-668进而参与miR-324/Mtfr或miR-668/Mtp18线粒体分裂调控途径,见图8。"
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