Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (8): 1172-1178.doi: 10.12307/2022.1000
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Jia Shengqi, Luo Wenlong, Tian Dingyuan, Zhang Xinhui, Cui Qian, Wang Chao, Pei Hanjun
Received:
2021-10-23
Accepted:
2021-12-28
Online:
2023-03-18
Published:
2022-07-27
Contact:
Pei Hanjun, MD, Chief physician, Department of Cardiology, The First Affiliated Hospital, Baotou Medical College of Inner Mongolia University of Science and Technology, Baotou 014010, Inner Mongolia Autonomous Region, China
About author:
Jia Shengqi, Master, Attending physician, Department of Cardiology, The First Affiliated Hospital, Baotou Medical College of Inner Mongolia University of Science and Technology, Baotou 014010, Inner Mongolia Autonomous Region, China
Supported by:
CLC Number:
Jia Shengqi, Luo Wenlong, Tian Dingyuan, Zhang Xinhui, Cui Qian, Wang Chao, Pei Hanjun. Expression of mitochondrial sirtuin 3 in mice with acute renal ischemia-reperfusion injury[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(8): 1172-1178.
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正常对照组的组织标本中未见明显的凋亡性改变;在假手术组凋亡水平很低,偶见单个凋亡细胞但与正常对照组存在差异(P < 0.05);缺血15 min再灌注组部分区域已有散在凋亡细胞出现,但总体凋亡水平仍较低,与假手术组比较差异明显(P < 0.01);缺血20 min再灌注组凋亡细胞数量有所增加,部分区域可见凋亡细胞聚集,每个聚集区域可见3-5个凋亡细胞,与缺血15 min再灌注组差异明显(P < 0.01);缺血25 min再灌注组凋亡细胞数量明显增加,凋亡细胞聚集频繁出现,且细胞聚集数目有所增加,与缺血20 min再灌注组比较差异明显(P < 0.01);缺血30 min再灌注组凋亡细胞大量出现,聚集趋势更为明显,同时部分区域可见正常的蓝色核数目减少,与缺血25 min再灌注组相比差异明显(P < 0.01),可见细胞凋亡水平随缺血时间延长而加重,见图2。 "
2.5 透射电镜下观察肾组织线粒体损伤情况 在透射电镜下放大20 000倍观察组织标本。正常对照组的线粒体超微结构正常,成簇排列在内质网周围,并且结构完好的线粒体大量存在。假手术组线粒体超微结构完整,整齐排列;缺血15 min再灌注组部分线粒体片段化,裂解,结构开始出现崩塌,但总体结构完整,且排列整齐;缺血20 min再灌注组可见部分区域内质网出现扩张,线粒体嵴部消失,肿胀、碎裂的情况更多见,部分线粒体完全崩解,且排列出现松散的趋势;缺血25 min再灌注组可见大量线粒体发生碎裂、崩解,几乎没有结构完整的线粒体保留,且线粒体的排列变得松散无序,内质网结构逐渐消失;缺血30 min再灌注组内质网结构崩塌,线粒体碎片化,结构消失,完全裂解,且正常线粒体数量大量减少,原有正常排列消失,见图3。"
2.6 肾组织线粒体分离鉴定结果 按照试剂盒说明书要求,对组织标本进行线粒体分离操作。用Western blot法分别检测分离前后GAPDH、Calnexin、mt-COX1、β-actin蛋白的表达,见图4,计算其在线粒体组分以及胞浆组分中的蛋白灰度值,随后按照线粒体组分灰度/(线粒体组分灰度+胞浆组分灰度),计算线粒体组分蛋白含量比例。按照分离要求,GAPDH≤15%、Calnexin≤15%、mt-COX1≥60%、β-actin≤15%即为分离成功。结果显示,线粒体组分蛋白含量比例:GAPDH为(7.84±1.35)%、Calnexin为(12.61±1.32)%、mt-COX1为(74.95±6.95)%、β-actin为(12.8±1.46)%,见图5,各组数据有显著性差异(P < 0.05),各蛋白比例均符合要求,线粒体分离完成。"
2.8 Western blot检测去乙酰化酶3、线粒体动力相关蛋白1、线粒体融合蛋白1蛋白水平 2.8.1 线粒体融合蛋白1蛋白水平 正常对照组线粒体融合蛋白1蛋白表达水平与假手术组比较差异无显著性意义(P > 0.05);缺血15 min再灌注组线粒体融合蛋白1蛋白表达水平与正常对照组和假手术组比较有所升高(P < 0.05);缺血20 min再灌注组线粒体融合蛋白1蛋白表达水平明显高于正常对照组和假手术组(P < 0.05),稍高于缺血15 min再灌注组,但差异无显著性意义(P > 0.05);缺血25 min再灌注组线粒体融合蛋白1蛋白表达水平与正常对照组、假手术组、缺血15,20 min再灌注组相比均呈现显著升高(P < 0.05);缺血30 min再灌注组线粒体融合蛋白1蛋白表达水平较缺血25 min再灌注组略微升高,但差异无显著性意义(P > 0.05),而该指标较正常对照组、假手术组、缺血15,20 min再灌注组均显著升高(P < 0.05)。对线粒体融合蛋白1与缺血时间的关系进行整理,发现随着缺血时间延长,该蛋白总体呈现逐渐升高的趋势,除20-25 min之间出现明显上升,其余各时间段升高较缓和,见图6。"
2.8.2 线粒体动力相关蛋白1蛋白水平 正常对照组线粒体动力相关蛋白1蛋白表达水平与假手术组比较差异无显著性意义(P > 0.05)。缺血15 min再灌注组线粒体动力相关蛋白1蛋白表达水平明显高于正常对照组及假手术组(P < 0.05);缺血20 min再灌注组线粒体动力相关蛋白1蛋白表达水平较缺血15 min再灌注组有所下降,但相比于正常对照组和假手术组升高(P < 0.05);缺血25 min再灌注组线粒体动力相关蛋白1蛋白表达水平较正常对照组及假手术组升高(P < 0.05),但与前两组相比有所下降(P < 0.05);缺血30 min再灌注组线粒体动力相关蛋白1蛋白表达水平处于缺血20 min及缺血25 min组之间,与上述两组分别比较,差异无显著性意义(P > 0.05),但与其余各组比较差异均有显著性意义(P < 0.05)。对线粒体动力相关蛋白1与缺血时间的关系进行整理,结果显示随着缺血时间延长,蛋白总体呈现先升高后降低趋势,于缺血15 min时出现高峰,随后下降,于30 min时仍未恢复正常,见图7。"
2.8.3 去乙酰化酶3蛋白水平 正常对照组与假手术组的去乙酰化酶3蛋白水平较低,两组间差异无显著性意义(P > 0.05);缺血15 min再灌注组去乙酰化酶3蛋白水平较正常对照组及假手术组略降低,差异无显著性意义(P > 0.05);缺血20 min再灌注组去乙酰化酶3蛋白水平与正常对照组和假手术组的水平相似(P > 0.05),但高于缺血15 min再灌注组(P < 0.05);缺血25 min再灌注组去乙酰化酶3蛋白水平与正常对照组、假手术组、缺血15 min再灌注组比较,均呈现明显升高(P < 0.05);缺血30 min再灌注组去乙酰化酶3表达进一步升高,且较其他组差异有显著性意义(P < 0.05)。对去乙酰化酶3与缺血时间的关系进行整理,发现随着缺血时间延长,蛋白总体呈现升高趋势,而缺血25,30 min时尤其明显,见图8。"
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