低温样品制备的小鼠心肌组织超微结构及其三维重构

生命科学的研究在微观水平和宏观水平两个方向上不断拓展。微观水平在结构生物学为手段解析蛋白质等生物大分子原子分辨率的三维结构，宏观水平则在构造生物学阶段研究机体的整体生命现象。观察以细胞为基本单位的整体生命现象，研究者首先要考虑的是尽可能还原样品在体内的生理状态，以及利用高分辨率大尺度的手段得到细胞乃至组织的空间分布。

常规生物电镜制样以化学固定为主，常温化学固定操作方便，但固定速率慢，容易引起细胞精细结构的失真，尤其是高放大倍数下细胞超微结构的变化较大。冷冻固定的速率快、保真程度高，能更好地接近细胞在生活时的状态。缺点是易产生冰晶对细胞造成损伤，操作难度大。目前，以冷冻固定为基础的低温电镜制样技术结合常规的树脂渗透、包埋技术能很好地弥补常温化学制样技术的一些缺陷，还能最大限度保持超微结构的精细程度。

心肌组织是一类典型的肌肉结构，因其自律性和收缩-舒张的特性有重要的研究意义，心肌细胞的超微结构对心肌功能研究有重要的作用。目前的心肌电镜样品大多数是临床医学样品，使用的是传统化学固定，容易造成人工伪影，细胞的结构也不够精细。本研究使用了新型的低温样品制备技术获得了野生C57小鼠心肌样品的电镜样品，并与常温化学固定方法进行了结构对比，在透射电镜下观察到了更加清楚和精细的细胞器及大分子物质，为此种新型制样技术研究哺乳动物超微结构提供了参考，也为探究心肌细胞的生理功能提供了基础。

本研究还在不同年龄阶段的野生C57小鼠心肌细胞中观察到了奇特的细胞核-线粒体互助现象。为了深入探究这一现象，本研究应用了电镜三维重构技术中的电子断层扫描技术及序列块表面扫描成像技术，获得了小鼠心肌组织的大尺度连续切片，并对心肌细胞中的线粒体及细胞核进行了三维重构，得到了核-线粒体相互作用的三维立体空间结构，与前期的二维电镜结果相作证。同时，本研究针对扫描电镜成像制备出一种适用于体扫描电镜的低温制样技术，改善了传统体电镜制样存在的结构收缩、重金属沉淀、人工伪像等问题，从而为三维体电镜技术的开发和应用提供技术上的参考。

针对细胞核-线粒体互作的机制探究，本研究使用了免疫荧光标记和Western blot实验定性地分析了与细胞凋亡、有丝分裂及去核化相关的特异性蛋白，结果表明，在小鼠心肌发育过程中核-线粒体的互作作用与线粒体融合蛋白Mitofusion2、丙酮酸脱氢酶PDC的变量有关，证明小鼠发育过程心肌细胞核不是一直都存在，会因为衰退被线粒体清除。

[1] RUSKA E, KNOLL M. Die magnetische Sammelspule für schnelle Elektronenstrahlen[J]. Themagnetic concentrating coil for fast electron beams.) Z. techn. Physik, 1931, 12: 389-400.
[2] INKSON B J. Scanning electron microscopy (SEM) and transmission electron microscopy(TEM) for materials characterization[M]//Materials characterization using nondestructive evaluation(NDE) methods. Elsevier, 2016: 17-43.
[3] 李伯勤, 张圣明. 医学超微结构基础[M]. 医学超微结构基础, 2003.
[4] 黄兰友, 刘绪平. 电子显微镜与电子光学[M]. 电子显微镜与电子光学, 1991.
[5] KRIVANEK O L, LOVEJOY T C, DELLBY N, et al. Vibrational spectroscopy in the electronmicroscope[J]. Nature, 2014, 514(7521): 209-212.
[6] 丁明孝, 梁凤霞, 洪健. 生命科学中的电子显微镜技术[M]. 北京：高等教育出版社, 2020.
[7] SUN C, MULLER E, MEFFERT M, et al. On the progress of scanning transmission electronmicroscopy (STEM) imaging in a scanning electron microscope[J]. Microscopy and Microanalysis,2018, 24(2): 99-106.
[8] YIN C C. Structural biology revolution led by technical breakthroughs in cryo-electron microscopy[J]. Chinese Physics B, 2018, 27(5): 058703.
[9] HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 forgenome engineering[J]. Cell, 2014, 157(6): 1262-1278.
[10] 张晓凯, 张丛丛, 刘忠民, 等. 冷冻电镜技术的应用与发展[J]. 科学技术与工程, 2019, 19(24): 9.
[11] ANAPATH. electron microscope[Z]. 2023.
[12] WILLIAMS D B, CARTER C B, WILLIAMS D B, et al. The transmission electron microscope[M]. Springer, 1996.
[13] HARRIS J R. Transmission electron microscopy in molecular structural biology: a historicalsurvey[J]. Archives of biochemistry and biophysics, 2015, 581: 3-18.
[14] KNOLL M. Aufladepotentiel und sekundäremission elektronenbestrahlter körper[J]. Zeitschriftfür technische Physik, 1935, 16(11): 467-475.
[15] ZWORYKIN V K. The scanning electron microscope[J]. Scientific American, 1942, 167(3):111-113.
[16] EVERHART T E, THORNLEY R. Wide-band detector for micro-microampere low-energyelectron currents[J]. Journal of scientific instruments, 1960, 37(7): 246.
[17] BOGNER A, JOUNEAU P H, THOLLET G, et al. A history of scanning electron microscopydevelopments: Towards “wet-STEM”imaging[J]. Micron, 2007, 38(4): 390-401.
[18] OATLEY C W. The early history of the scanning electron microscope[J]. Journal of AppliedPhysics, 1982, 53(2): R1-R13.
[19] 田中敬一, 永谷隆. 图解扫描电子显微镜: 生物样品制备[M]. 图解扫描电子显微镜: 生物样品制备, 1984.
[20] 张大同. 扫描电镜与能谱仪分析技术[M]. 扫描电镜与能谱仪分析技术, 2009.
[21] 施明哲. 扫描电镜和能谱仪的原理与实用分析技术[M]. 扫描电镜和能谱仪的原理与实用分析技术, 2015.
[22] BUCKLEY A. JI Goldstein, DE Newbury, P. Echlin, DC Joy, AD RomigJr, CE Lyman, C. FioriE. Lifshin 1992. Scanning Electron Microscopy and X-Ray Microanalysis. A Text for Biologists,Materials Scientists, and Geologists, xviii+ 820 pp. New York, London: Plenum Press. ISBN 0306 44175 6.[J]. Geological Magazine, 1993, 130(3): 402-403.
[23] GOLDSTEIN J I, NEWBURY D E, ECHLIN P, et al. Special topics in electron beam x-raymicroanalysis[J]. Scanning Electron Microscopy and X-ray Microanalysis: Third Edition, 2003:453-536.
[24] VON ARDENNE M. Das Elektronen-Rastermikroskop: Theoretische Grundlagen[J].Zeitschrift Physik, 1938, 109(9-10): 553-572.
[25] CLAESSON S, SVENSSON A. A new ultramicrotome for electron microscopy[J]. ExperimentalCell Research, 1956, 11(1): 105-114.
[26] ADRIAN M, DUBOCHET J, FULLER S D, et al. Cryo-negative staining[J]. Micron, 1998, 29(2-3): 145-160.
[27] AEBI U, COHN J, BUHLE L, et al. The nuclear lamina is a meshwork of intermediate-typefilaments[J]. Nature, 1986, 323(6088): 560-564.
[28] BRENNER S, HORNE R. A negative staining method for high resolution electron microscopyof viruses[J]. Biochimica et biophysica acta, 1959, 34: 103-110.
[29] NAGANO T. Fixation 1. Chemical Fixation[J]. Kenbikyo, 2000, 35.
[30] HENDRICKS G M. Metal shadowing for electron microscopy[J]. Electron Microscopy: Methodsand Protocols, 2014: 73-93.
[31] HEUSER J E. The origins and evolution of freeze-etch electron microscopy[J]. Journal ofelectron microscopy, 2011, 60(suppl_1): S3-S29.
[32] HIROKAWA N. Quick-freeze, deep-etch electron microscopy.[J]. Journal of Electron Microscopy,1989, 38 Suppl: S123.
[33] HEUSER J. Quick-freeze, deep-etch preparation of samples for 3-D electron microscopy[J].Trends in Biochemical Sciences, 1981, 6: 64-68.
[34] BOZZOLA J J. Conventional specimen preparation techniques for scanning electron microscopyof biological specimens[J]. Electron microscopy: methods and protocols, 2007: 449-466.
[35] HALL C E. Visualization of individual macromolecules with the electron microscope[M]. NationalAcad Sciences, 1956.
[36] KUO J. Electron microscopy: methods and protocols: volume 369[M]. Springer Science &Business Media, 2008.
[37] CRANG R, KLOMPARENS K L. Artifacts in biological electron microscopy[M]. Artifacts inbiological electron microscopy, 1988.
[38] MOOR H, MÜHLETHALER K. Fine structure in frozen-etched yeast cells[J]. The Journal ofcell biology, 1963, 17(3): 609-628.
[39] NERMUT M, EASON P. Cryotechniques in macromolecular research (a comparative study).[J]. Scanning microscopy. Supplement, 1989, 3: 213-24.
[40] MCDONALD K, MÜLLER-REICHERT T. Cryomethods for thin section electron microscopy[J]. Methods in enzymology, 2002, 351: 96-123.
[41] MÜLLER-REICHERT T, HOHENBERG H, O’TOOLE E, et al. Cryoimmobilization and threedimensionalvisualization of C. elegans ultrastructure[J]. Journal of microscopy, 2003, 212(1):71-80.
[42] FERNÁNDEZ-MORÁN H. Low temperature preparation techniques for electron microscopyof biological specimens based on rapid freezing with liquid helium II[J]. Annals of the NewYork Academy of Sciences (US), 1960, 85.
[43] MOOR H. Theory and Practice of High Pressure Freezing[J]. Springer Berlin Heidelberg, 1987.
[44] VAN HARREVELD A, CROWELL J. Electron microscopy after rapid freezing on a metalsurface and substitution fixation[J]. The Anatomical Record, 1964, 149(3): 381-385.
[45] HIPPE-SANWALD S. Impact of freeze substitution on biological electron microscopy[J]. Microscopyresearch and technique, 1993, 24(5): 400-422.
[46] MOOR H, RIEHLE U. Snap-Freezing Under High Pressure: A New Fixation Technique forFreeze-Etching[J]. Micron, 1968, 2: 33.
[47] HUMBEL B, MÜLLER M. Freeze substitution and low temperature embedding[J]. ScanningElectron Microscopy, 1985, 4(1): 19.
[48] NICOLAS M T, BASSOT J M. Freeze substitution after fast-freeze fixation in preparation forimmunocytochemistry[J]. Microscopy research and technique, 1993, 24(6): 474-487.
[49] KELLENBERGER E. The response of biological macromolecules and supramolecular structuresto the physics of specimen cryopreparation[J]. Cryotechniques in biological electron microscopy,1987: 35-63.
[50] WHALLEY E. Scheiner’s halo: evidence for ice Ic in the atmosphere[J]. Science, 1981, 211(4480): 389-390.
[51] SHIMONI E, MÜLLER M. On optimizing high-pressure freezing: from heat transfer theory toa new microbiopsy device.[J]. Journal of microscopy, 1998, 192(Pt 3): 236-247.
[52] STEINBRECHT R A, MüLLER M. Freeze-Substitution and Freeze-Drying[Z]. 1987.
[53] KRIJNSE LOCKER J, SCHMID S L. Integrated electron microscopy: super-duper resolution[J]. PLoS biology, 2013, 11(8): e1001639.
[54] MCDONALD K L, AUER M. High-pressure freezing, cellular tomography, and structural cellbiology[J]. Biotechniques, 2006, 41(2): 137-143.
[55] KEENE D R, TUFA S F. Connective Tissue Ultrastructure: A Direct Comparison betweenConventional Specimen Preparation and High-Pressure Freezing/Freeze-Substitution[J]. TheAnatomical Record, 2020, 303(6): 1514-1526.
[56] VARSANO N, WOLF S G. Electron microscopy of cellular ultrastructure in three dimensions[J]. Current opinion in structural biology, 2022, 76: 102444.
[57] SAIBIL H R. Cryo-EM in molecular and cellular biology[J]. Molecular Cell, 2022, 82(2):274-284.
[58] TURK M, BAUMEISTER W. The promise and the challenges of cryo-electron tomography[J].FEBS letters, 2020, 594(20): 3243-3261.
[59] DENK W, HORSTMANN H. Serial block-face scanning electron microscopy to reconstructthree-dimensional tissue nanostructure[J]. PLoS biology, 2004, 2(11): e329.
[60] PEDDIE C J, COLLINSON L M. Exploring the third dimension: volume electron microscopycomes of age[J]. Micron, 2014, 61: 9-19.
[61] WEBB R I, SCHIEBER N L. Volume scanning electron microscopy: serial block-face scanningelectron microscopy focussed ion beam scanning electron microscopy[J]. Cellular Imaging:electron tomography and related techniques, 2018: 117-148.
[62] ZHENG Z, LAURITZEN J S, PERLMAN E, et al. A complete electron microscopy volume ofthe brain of adult Drosophila melanogaster[J]. Cell, 2018, 174(3): 730-743.
[63] PARAJULI L K, KOIKE M. Three-dimensional structure of dendritic spines revealed by volumeelectron microscopy techniques[J]. Frontiers in Neuroanatomy, 2021, 15: 627368.
[64] SCHNEIDER J P, HEGERMANN J, WREDE C. Volume electron microscopy: analyzing thelung[J]. Histochemistry and Cell Biology, 2021, 155: 241-260.
[65] TAYLOR K A, GLAESER R M. Electron diffraction of frozen, hydrated protein crystals[J].Science, 1974, 186(4168): 1036-1037.
[66] DUBOCHET J, MCDOWALL A. Vitrification of pure water for electron microscopy[J]. Journalof Microscopy, 1981, 124(3): 3-4.
[67] WOLF S G, HOUBEN L, ELBAUM M. Cryo-scanning transmission electron tomography ofvitrified cells[J]. Nature methods, 2014, 11(4): 423-428.
[68] BÄUERLEIN F J, BAUMEISTER W. Towards visual proteomics at high resolution[J]. Journalof Molecular Biology, 2021, 433(20): 167187.
[69] TITZE B, GENOUD C. Volume scanning electron microscopy for imaging biological ultrastructure[J]. Biology of the Cell, 2016, 108(11): 307-323.
[70] SMITH D, STARBORG T. Serial block face scanning electron microscopy in cell biology:Applications and technology[J]. Tissue and Cell, 2019, 57: 111-122.
[71] DEERINCK T J, SHONE T, BUSHONG E A, et al. High-performance serial block-face SEMof nonconductive biological samples enabled by focal gas injection-based charge compensation[J]. Journal of microscopy, 2018, 270(2): 142-149.
[72] SCHWARTZ C L, HEUMANN J M, DAWSON S C, et al. A detailed, hierarchical study of Giardialamblia’s ventral disc reveals novel microtubule-associated protein complexes[M]. PublicLibrary of Science San Francisco, USA, 2012.
[73] SEVERS N J. The cardiac muscle cell[J]. Bioessays, 2000, 22(2): 188-199.
[74] WALKER C A, SPINALE F G. The structure and function of the cardiac myocyte: a reviewof fundamental concepts[J]. The Journal of thoracic and cardiovascular surgery, 1999, 118(2):375-382.
[75] CUI Y, ZHENG Y, LIU X, et al. Single-cell transcriptome analysis maps the developmentaltrack of the human heart[J]. Cell reports, 2019, 26(7): 1934-1950.
[76] PINNELL J, TURNER S, HOWELL S. Cardiac muscle physiology[J]. Continuing Educationin Anaesthesia Critical Care & Pain, 2007, 7(3): 85-88.
[77] HUNTER P J, MCCULLOCH A D, TER KEURS H. Modelling the mechanical properties ofcardiac muscle[J]. Progress in biophysics and molecular biology, 1998, 69(2-3): 289-331.
[78] WOODCOCK E A, MATKOVICH S J. Cardiomyocytes structure, function and associatedpathologies[J]. The international journal of biochemistry & cell biology, 2005, 37(9): 1746-1751.
[79] SPACH M S, KOOTSEY J M. The nature of electrical propagation in cardiac muscle[J]. AmericanJournal of Physiology-Heart and Circulatory Physiology, 1983, 244(1): H3-H22.
[80] WIKIPEDIA. Cardiac Muscle[J/OL]. Wikimedia Foundation. https://en.wikipedia.org/wiki/Cardiac_muscle.
[81] IOVINE J C, CLAYPOOL S M, ALDER N N. Mitochondrial compartmentalization: emergingthemes in structure and function[J]. Trends in biochemical sciences, 2021, 46(11): 902-917.
[82] BARZDA V, GREENHALGH C, DER AU J A, et al. Visualization of mitochondria in cardiomyocytes[J]. Optics express, 2005, 13(20): 8263-8276.
[83] PRINCE F P. Lamellar and tubular associations of the mitochondrial cristae: unique forms ofthe cristae present in steroid-producing cells[J]. Mitochondrion, 2002, 1(4): 381-389.
[84] POHJOISMÄKI J L, GOFFART S. The role of mitochondria in cardiac development and protection[J]. Free Radical Biology and Medicine, 2017, 106: 345-354.
[85] ADANIYA S M, O-UCHI J, CYPRESS M W, et al. Posttranslational modifications of mitochondrialfission and fusion proteins in cardiac physiology and pathophysiology[J]. AmericanJournal of Physiology-Cell Physiology, 2019, 316(5): C583-C604.
[86] PARK M K, ASHBY M C, ERDEMLI G, et al. Perinuclear, perigranular and sub-plasmalemmalmitochondria have distinct functions in the regulation of cellular calcium transport[J]. TheEMBO journal, 2001, 20(8): 1863-1874.
[87] HOLLANDER J M, THAPA D, SHEPHERD D L. Physiological and structural differences inspatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies[J].American Journal of Physiology-Heart and Circulatory Physiology, 2014, 307(1): H1-H14.
[88] LU X, THAI P N, LU S, et al. Intrafibrillar and perinuclear mitochondrial heterogeneity in adultcardiac myocytes[J]. Journal of molecular and cellular cardiology, 2019, 136: 72-84.
[89] BRIELER J, BREEDEN M A, TUCKER J. Cardiomyopathy: an overview[J]. American familyphysician, 2017, 96(10): 640-646.
[90] GOODWIN J. The frontiers of cardiomyopathy.[J]. British heart journal, 1982, 48(1): 1.
[91] JEFFERIES J L, TOWBIN J A. Dilated cardiomyopathy[J]. The Lancet, 2010, 375(9716):752-762.
[92] MARON B J, BONOW R O, CANNON III R O, et al. Hypertrophic cardiomyopathy[J]. NewEngland Journal of Medicine, 1987, 316(14): 844-852.
[93] KUSHWAHA S S, FALLON J T, FUSTER V. Restrictive cardiomyopathy[J]. New EnglandJournal of Medicine, 1997, 336(4): 267-276.
[94] CORRADO D, LINK M S, CALKINS H. Arrhythmogenic right ventricular cardiomyopathy[J]. New England journal of medicine, 2017, 376(1): 61-72.
[95] BOCK F J, TAIT S W. Mitochondria as multifaceted regulators of cell death[J]. Nature reviewsMolecular cell biology, 2020, 21(2): 85-100.
[96] ANNESLEY S J, FISHER P R. Mitochondria in health and disease[J]. Cells, 2019, 8(7): 680.
[97] MACDONALD R, BARNES K, HASTINGS C, et al. Mitochondrial abnormalities in Parkinson’sdisease and Alzheimer’s disease: can mitochondria be targeted therapeutically?[J]. BiochemicalSociety Transactions, 2018, 46(4): 891-909.
[98] MEYERS D E, BASHA H I, KOENIG M K. Mitochondrial Cardiomyopathy.[J]. Texas HeartInstitute Journal, 2013, 40(4).
[99] PROTASONI M, ZEVIANI M. Mitochondrial structure and bioenergetics in normal and diseaseconditions[J]. International Journal of Molecular Sciences, 2021, 22(2): 586.
[100] KLOPSTOCK T, PRIGLINGER C, YILMAZ A, et al. Mitochondrial Disorders.[J]. DeutschesAerzteblatt International, 2021, 118(44).
[101] CHINNERY P F, TURNBULL D M. Epidemiology and treatment of mitochondrial disorders[J]. American journal of medical genetics, 2001, 106(1): 94-101.
[102] ZEVIANI M, MORAES C T, DIMAURO S, et al. Deletions of mitochondrial DNA in Kearns-Sayre syndrome[J]. Neurology, 1988, 38(9): 1339-1339.
[103] CHOMYN A, MARTINUZZI A, YONEDA M, et al. MELAS mutation in mtDNA binding sitefor transcription termination factor causes defects in protein synthesis and in respiration but nochange in levels of upstream and downstream mature transcripts.[J]. Proceedings of the NationalAcademy of Sciences, 1992, 89(10): 4221-4225.
[104] CRIMI M, PAPADIMITRIOU A, GALBIATI S, et al. A new mitochondrial DNA mutation inND3 gene causing severe Leigh syndrome with early lethality[J]. Pediatric research, 2004, 55(5): 842-846.
[105] JOHNSON M, TURNBULL D, DICK D, et al. A partial deficiency of cytochrome c oxidasein chronic progressive external ophthalmoplegia[J]. Journal of the neurological sciences, 1983,60(1): 31-53.
[106] RANJBARVAZIRI S, KOOIKER K B, ELLENBERGER M, et al. Altered cardiac energeticsand mitochondrial dysfunction in hypertrophic cardiomyopathy[J]. Circulation, 2021.
[107] LI J, LI J, CHEN Y, et al. The Role of Mitochondria in Metabolic Syndrome–Associated Cardiomyopathy[J]. Oxidative Medicine and Cellular Longevity, 2022, 2022.
[108] LIU S, BAI Y, HUANG J, et al. Do mitochondria contribute to left ventricular non-compactioncardiomyopathy? New findings from myocardium of patients with left ventricular noncompactioncardiomyopathy[J]. Molecular Genetics and Metabolism, 2013, 109(1): 100-106.
[109] ARBUSTINI E, DIEGOLI M, FASANI R, et al. Mitochondrial DNA mutations and mitochondrialabnormalities in dilated cardiomyopathy[J]. The American journal of pathology, 1998,153(5): 1501-1510.
[110] RUSSELL O M, GORMAN G S, LIGHTOWLERS R N, et al. Mitochondrial diseases: hopefor the future[J]. Cell, 2020, 181(1): 168-188.
[111] COLLINS H E, KANE M S, LITOVSKY S H, et al. Mitochondrial morphology and mitophagyin heart diseases: qualitative and quantitative analyses using transmission electron microscopy[J]. Frontiers in Aging, 2021, 2: 670267.
[112] TASKAEVA Y S, BGATOVA N, SAVCHENKO S, et al. Ultrastructure of Endothelial Cellsof Myocardial Capillaries in Burn Septicotoxemia[J]. Bulletin of Experimental Biology andMedicine, 2021, 171: 393-398.
[113] TSOUPRI E, KOSTAVASILI I, KLOUKINA I, et al. Myospryn deficiency leads to impairedcardiac structure and function and schizophrenia-associated symptoms[J]. Cell and Tissue Research,2021, 385(3): 675-696.
[114] ALI E M, EL-SAYED S M, ELBASTAWISY Y M. Ultrastructural aberrations, histological disruptionand upregulation of the VEGF, CD34 and ASMA immunoexpression in the myocardiumof anemic albino rats[J]. Acta Histochemica, 2021, 123(5): 151731.
[115] 徐凡, 张艳美. 不同组织匀浆法对心肌组织线粒体提取质量的影响[J]. 汕头大学医学院学报, 2021, 34(2): 4.
[116] 白静, 张文丽, 张军伟, 等. 脓毒症大鼠心肌氧化应激损伤及心肌细胞超微结构变化[J]. 细胞与分子免疫学杂志, 2015(5): 5.
[117] 常红波, 王振涛, 吴鸿. 扩张型心肌病大鼠心肌细胞亚细胞器超微结构分析[J]. 国际心血管病杂志, 2021, 48(5): 4.
[118] 武迎, 陈东, 方微, 等. 68 例大心脏移植的受体心脏病理学特征分析[J]. 心肺血管病杂志,2017(005): 036.
[119] 孙洋, 赵红. 致心律失常性心肌病受体心脏病理形态学及超微结构[J]. 临床与病理杂志,2017, 37(8): 6.
[120] HOFFMAN H, GRIGG G. An electron microscopic study of mitochondria formation[J]. ExperimentalCell Research, 1958, 15(1): 118-131.
[121] MORI H. Electron microscopic studies of the ascites tumor cells. Report 1. On the mitochondriawithin the nuclei of various tumor cells and regenerating liver cells of the newt[J]. FukushimaJ Med Sci, 1960, 7: 21-32.
[122] BRANDES D, SCHOFIELD B, SLUSSER R, et al. Studies of L1210 Leukemia. I. Ultrastructureof Solid and Ascites Cells[J]. JNCI Journal of the National Cancer Institute, 1966, 37(4):467-485.
[123] KLUG H. On the occurrence of mitochondria in the cell nucleus[J]. Die Naturwissenschaften,1966, 53(13): 339.
[124] BLOOM G D. A nucleus with cytoplasmic features[J]. The Journal of Cell Biology, 1967, 35(1): 266.
[125] OLIVA H, VALLE A, FLORES L D, et al. Intranuclear mitochondriae in Hodgkin’s disease[J]. Virchows Archiv B, 1972, 12: 189-194.
[126] SCHUMACHER H, SZEKELY I, PATEL S, et al. Leukemic mitochondria: I. Acute myeloblasticleukemia[J]. The American Journal of Pathology, 1974, 74(1): 71.
[127] JENSEN H, ENGEDAL H, SAETERSDAL T S. Ultrastructure of mitochondria-containingnuclei in human myocardial cells[J]. Virchows Archiv B, 1976, 21(1): 1-12.
[128] TSYPLENKOVA V, BESKROVNOVA N. The comparative morphological and morphometriccharacteristics of the myocardium in patients with a clinical diagnosis of hypertrophic cardiomyopathy[J]. Arkhiv patologii, 1993, 55(3): 26-29.
[129] TAKEMURA G, TAKATSU Y, SAKAGUCHI H, et al. Intranuclear mitochondria in humanmyocardial cells.[J]. Pathology, research and practice, 1997, 193(4): 305-311.
[130] BAKEEVA L, SKULACHEV V, SUDARIKOVA Y V, et al. Mitochondria enter the nucleus(one further problem in chronic alcoholism)[J]. Biochemistry (Moscow), 2001, 66: 1335-1341.
[131] FIDZIAŃSKA A, BILIŃSKA Z T, TESSON F, et al. Obliteration of cardiomyocyte nucleararchitecture in a patient with LMNA gene mutation[J]. Journal of the neurological sciences,2008, 271(1-2): 91-96.
[132] ELDAROV C M, VANGELY I M, VAYS V B, et al. Mitochondria in the nuclei of rat myocardialcells[J]. Cells, 2020, 9(3): 712.
[133] ZERVOPOULOS S D, BOUKOURIS A E, SALEME B, et al. MFN2-driven mitochondria-tonucleustethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvatedehydrogenase complex[J]. Molecular Cell, 2022, 82(5): 1066-1077.
[134] ZHAO B, MEI Y, SCHIPMA M J, et al. Nuclear condensation during mouse erythropoiesisrequires caspase-3-mediated nuclear opening[J]. Developmental cell, 2016, 36(5): 498-510.