【作者】 许莹；

【导师】 田虎；

【作者基本信息】 山东大学 ， 普外科（专业学位）， 2019， 博士

【摘要】 目的:Telocytes细胞(TCs)是一种具有极长、极薄端足(TPS)的新型间质细胞。自从Popescu等人[1]率先将这种特殊的间质细胞命名为“Telocytes”以来,为了与其他间质细胞类型进行区分(例如成纤维细胞、纤维母细胞样细胞、间充质细胞等),越来越多的研究人员已经发现Telocytes细胞存在于人和实验哺乳动物的各种腔隙性和非空腔性脏器中。在随后的七到八十年里,此种细胞的存在、性质和功能在不同的科学团队中引起了很大的争议。到1960年,Taxi和他的团队通过应用透视电镜技术对此种特殊细胞进行观察后得出了此细胞区别于间质细胞(ICC)如神经元细胞、“施万”细胞、平滑肌细胞、成纤维细胞和巨噬细胞等的一种新的细胞类型。从Telocyte细胞胞体发出的telopode是区别于神经元间质细胞的典型结构,也是与同源性和异源性细胞相互连接的“桥梁”,大量的Telocyte细胞在组织和器官中形成三维网格状结构。Telocytes在不同的组织器官内表现出不同的功能,并参与疾病的发生发展。如Telocytes通过对同源或异源性细胞之间接触的连接形成3D网络的结构从而在整个组织中起到机械支撑的作用。观察成人神经肌肉接头处,Telocytes存在于囊膜的内层和最外层,可通过其特殊的微环境,为控制肌肉张力和运动活动提供机械支持。Telocytes在组织中形成的三维网络中起到的另一个重要作用是主动传递细胞间信号,在不同的组织器官中Telocytes可以建立直接的细胞与细胞之间的信号传导通路,如形成出芽的小泡、胞外体或其他微小分子结合而成的外分泌体和/或旁分泌体。Telocyte细胞通过传递细胞信息的方式调节、控制和参与组织器官的损伤、修复、再生和凋亡过程。为了明确Telocyte细胞在敲基因小鼠体内存在的证据和可能表达的免疫组化指标,同时探索Telocyte细胞是否参与动脉粥样硬化血管损伤修复的过程,本实验设计过程中涉及到了两部分的内容,分别对以上两个问题进行研究。方法:首先随机选取ApoE-/-小鼠,将小鼠的心脏,肾脏和肝脏等新鲜组织用通用组织固定液进行标本固定后行免疫组化和免疫荧光实验。免疫组化生物学指标选取CD34,CD28,CD117,PDGFR和Vimentin,免疫荧光生物学指标选取CD34/CD117,CD34/PDGFR和CD28/Vimentin双染指标。通过两种免疫生物学方法寻找Telocyte细胞存在于ApoE-/-小鼠重要器官中的证据,并确定Telocyte细胞的形态结构和在组织器官中的分布位置,初步明确telopode生物学表达指标。然后,随机选取ApoE-/-小鼠和正常野生型小鼠各40只,经含有21%脂肪和0.15%胆固醇的高脂饲料喂食12周后进行小鼠体内大动脉血管的粥样硬化斑块的测量,并将ApoE-/-小鼠的颈动脉斑块面积占整体颈动脉面积不小于45%,同时将主动脉根部的血管壁增厚,管腔变窄作为动物模型建成的标志。将建成的动物模型和正常小鼠同时进行颈动脉血管的部分离断损伤和血管损伤的吻合术,术后需确保吻合血管的血流通畅性。麻醉复苏后根据时间随机将小鼠放置于标准饲养箱内,所有术后小鼠皆给与相同的饲养方式,两组小鼠分别于术后8小时,术后48小时和术后21天取颈动脉损伤修复血管作为下一步研究标本,所有标本取出后放入通用组织固定液中固定24小时同时进行相应编号。所有标本都进行免疫组化实验,免疫荧光实验和细胞增殖实验。为了观察Telocyte细胞在动脉粥样硬化血管中的形态结构和超微细胞器的改变,从形成动脉粥样硬化的ApoE-/-小鼠颈动脉血管中应用透射电镜的实验方法进行查找和验证。同时首次应用ApoE-/-小鼠正常颈动脉血管活体组织进行Telocyte细胞的原代细胞培养,在高倍电子显微镜下观察Telocyte细胞的生物学特性。结果:Telocyte细胞在心肌组织中分布广泛,对CD34,CD117及PDGFR三种免疫组化指标表达阳性,但不是所有的Telocyte细胞胞体和细胞发出的端足telopodes都对以上三种免疫组化指标阳性表达,其中,CD34染色可阳性染色Telocyte细胞的胞体和所有的telopodes,且通过CD34的染色可以发现Telocyte细胞分布于心肌组织间质内,从胞体发出的多条细长的telopodes端足与邻近的细胞关系紧密,多个Telocyte细胞可形成类似于网状结构的框架。CD117染色和PDGFR染色仅仅可使telopodes表达阳性,说明在telopodes内存在某种信号蛋白可表达CD117和PDGFR两种免疫生物学指标。CD28染色在心肌组织中表现出阴性结果,说明Telocyte细胞对CD28阴性表达。在肝脏组织实质中的中央小静脉附近,Telocyte细胞对CD34免疫组化指标阳性表达,而且可以看到Telocyte细胞分布在沿肝中央小静脉的肝实质内,其细胞胞体和从胞体发出的telopodes端足皆表达CD34,Telocyte细胞表现出蝶翼状、梭型等形状。肝血窦位于肝板之间,腔大而不规则,在肝“迪赛”间隙中也存在Telocyte细胞,此处的Telocyte细胞对CD117免疫组化指标部分表达,不能完整的展现出Telocyte细胞的胞体形状,仅表现出telopodes的走形和分布。在肝实质内也存在Telocyte细胞,telopodes对PDGFR免疫组化指标阳性表达。肾髓质和肾皮质内的Telocyte细胞对CD34免疫组化生物指标阳性染色,且Telocyte细胞分布在肾髓质的肾小球间质内和肾皮质的近曲小管和远曲小管上皮细胞之间。CD117和PDGFR两种免疫组化指标在肾组织中对Telocyte细胞阴性染色。免疫荧光双染指标的实验,在CD28/Vimentin染色中,心脏组织中的毛细血管壁及部分心肌细胞对CD28/Vimentin 阳性表达,但Telocyte对两种指标阴性表达;在CD34/CD117染色中,CD34阳性染色的Telocyte细胞明显可见,且telopodes也可表达,但CD117染色的Telocyte细胞表达不明确,不能分辨出明显的Telocyte细胞的形态结构和分布情况;在CD34/PDGFR染色中,心脏组织中的Telocyte细胞的形态结构和分布情况明显可见,Telocyte细胞的胞体对PDGFR部分表达,对CD34完全表达,CD34/PDGFR双染下的多个Telocyte细胞胞体和telopodes端足之间形成三维立体的网状结构存在于心脏间质中。免疫荧光双染指标CD34/PDGFR对ApoE-/-小鼠肝脏内肝中央小静脉及周围细胞进行染色,发现PDGFR对肝实质细胞普遍阳性染色,因此不能明确的分辨Telocyte细胞对PDGFR表达的性质。Telocyte细胞和中央静脉上皮细胞对CD34阳性染色,且Telocyte细胞分布在肝中央小静脉的周围。选取CD28/Vimentin,CD34/CD117和CD34/PDGFR三组双染色免疫荧光生物指标对ApoE-/-小鼠肝实质及肝血窦进行染色。肝细胞对CD2 8/Vimentin和CD34/CD117荧光标记普遍染色。在肝组织肝血窦及周围细胞染色中,CD34阳性表达的Telocyte细胞清晰可辨,PDGFR荧光染色对大部分肝实质细胞阳性表达。CD28/Vimentin对肾髓质内肾小球细胞普遍阳性染色。CD34阳性表达的Telocyte细胞存在于肾髓质相邻肾小球上皮细胞之间,但肾小球上皮细胞和Telocyte细胞对CD117和PDGFR阴性表达。在本实验的第二部分中,我们发现经过12周高脂饮食饲养的APoE-/-小鼠的大动脉壁内可形成典型的动脉粥样硬化改变。在ApoE-/-小鼠颈动脉损伤修复的不同阶段,出现了 Telocyte细胞不同的生物学改变:在血管损伤初期,Telocyte细胞开始聚集在管壁的断裂处,数量明显比正常的血管壁中膜内的细胞数量多,在修复的中期却出现了 Telocyte细胞的数量急剧减少的现象,而且Telocyte细胞参与整个血管壁修复的过程,在血管壁完全修复后可以见到Telocyte细胞的分布和数量与未损伤的血管壁内的Telocyte细胞的数量相同;在正常小鼠的对照实验中,可见Telocyte细胞的形态结构正常,在血管壁损伤修复的不同阶段出现了数量上从多到少再到正常的现象,而且血管修复的时间明显比ApoE-/-小鼠血管壁修复的时间短。通过透射电镜扫描技术可以清晰的观察到Telocyte细胞内部的超微结构。Telocyte细胞由胞体和telopode组成,胞体形状各异,可呈椭圆形,三角形和不规则形等,telopode的数量也各不相同,本实验中可以观察到的telopode的数量为1-5条。在动脉粥样硬化血管壁内的Telocyte细胞的超微结构开始发生改变,细胞核形态不规则,核膜变得模糊,体积缩小,内部异染色质开始出现,核仁形态不规则。Telocyte细胞质内的线粒体出现空泡变性,数量减少,分布不均匀,线粒体脊消失。粗面内质网数量减少。细胞质内出现大小不等的脂质体。Telopode的连续性终断,直径变粗,迂曲肿胀,其内部细胞器显示不清。Telocyte周围可见大量胶原纤维和弹力纤维。在损伤的颈动脉血管壁内的Telocyte细胞形态及结构发生明显的改变,胞质内的细胞器数量增多,尤其是高尔基体和粗面内质网的数量,细胞核内染色质浓聚,在透射电镜下呈现明显的大片状的暗区。细胞膜周围出现大量的出芽小泡,Tp内的细胞器数量也增多,串珠样的膨大处的直径较正常Telocyte细胞Tp膨大处的直径粗。1.结论:免疫生物学技术应用于Telocyte细胞的形态结构和半定量分析中,发现在ApoE-/-小鼠的心脏,肾脏和肝脏组织中Telocyte细胞存在不同表达的生物免疫标记物,其中CD34阳性表达是Telocyte细胞的特异性表现,可以出现在各种重要的组织器官中。2.Telocyte细胞和telopodes可以表达不一样的生物免疫标记物;在不同的组织器官中,Telocyte细胞表达的免疫标记物不尽相同。3.在同一组织的不同部位,Telocyte细胞表达的免疫标记物也不同。4.ApoEo-/-小鼠在经过12周的高脂饮食饲养后,大动脉血管壁可形成典型的粥样脂质斑块。5.Telocyte细胞存在于小鼠动脉血管壁的外膜层和中膜层,对CD34、CD117和PDGFR免疫标记物阳性表达,对CD28和Vimentin免疫标记物阴性表达;Telopode对PDGFR和CD34免疫标记物阳性表达。6.在小鼠动脉损伤修复的过程中,Telocyte参与组织修复的整个过程,在重建血管壁的过程中,Telocyte细胞起到了关键的作用;在动脉粥样硬化血管中的Telocyte细胞的形态结构和细胞器的超微结构皆发生改变,说明Telocyte细胞参与动脉粥样硬化病理过程的发生和发展,血管壁的损伤势必造成Telocyte细胞的改变。7.在小鼠正常的动脉血管中,Telocyte细胞内的细胞器清晰可见,细胞核结构正常,与telopode的细胞质相通;在组织发生病理性的改变过程中,Telocyte细胞也发生相应的改变。更多还原

【Abstract】 Objective:Telocytes(TCs)is a kind of extremely special and new mesenchymal cells.Since Popescu et al[1]take the lead in those mesenchymal cells named "Telocyte",more and more researchers have found that Telocytes exist in human beings and experimental mammals and distinguish from other stromal cell types(such as fibroblasts,fibroblast cells,mesenchymal cells,etc.).Over the next seven to eight decades,the existence,feature and function of such cell generated considerable controversy among different scientific groups.By the 1960s,Taxi had observed this particular cell type using transmitted electron microscopy,and had identified a new cell type that distinguished from interstitial cells(ICC)such as neurons,Schwan cells,smooth muscle cells,fibroblasts,and macrophages.The telopode oriented from the Telocyte’s body is a typical structure differenting from the neuronal interstitial cells and also a "bridge" connecting with homologous and heterogenous cells.A large number of Telocytes form a three-dimensional mesh-like structure in tissues and organs.Telocytes in different tissues and organs show distinct functions,and participate in the progress of diseases’development.Such as Telocytes through the contact between the same or different cells connect to form a 3D network structure which have the effect of mechanical support in the organs.At adult neuromuscular junction,Telocytes exist in the lining of the posterior capsule and the outermost layer,through its special micro-environment,providing mechanical support for the control muscle tension and sport activities.Telocytes play another important role in cell signals,being established between the cells and cell signal transductive process directly,such as the formation of budding vesicles,the combination of extracellular body or other small molecules of exocrine body and/or paracrine body.By means of transmitting cell information,Telocytes regulate,control and participate in the process of tissue and organ damage,repair,regeneration and apoptosis progresses.In order to determine the evidence of Telocytes in gene knockout mice and the immunohistochemical indexes of their possible expression,and to explore whether Telocytes are involved in the process of repairing atherosclerotic vascular injury,this experiment is divided into two parts.Methods:First,ApoE-/-mice were randomly selected.The hearts,kidneys,livers and other fresh tissues were immobilized with universal tissue fixative solution for immunohistochemical and immunofluorescence experiments.CD34,CD28,CD117,PDGFR and Vimentin bio-markers were selected and CD34/CD117,CD34/PDGFR and CD28/Vimentin double markers were selected for immunofluorescence biological indicators.Two immunobiological methods were used to find Telocytes in ApoE-/-mice.The morphological structure and distribution of Telocyte were determined,and the biological expression indexes of telopode were preliminarily determined.Forty ApoE-/-mice and forty normal wild-type mice were selected and fed the same high-fat diet,containing 21%fat and 0.15%cholesterol for 12 weeks.Atherosclerosis disease in the arteries of the mice was measured and the carotid plaque area of the mice should account for no less than 45%of the total carotid artery diameter,and the thickening of the vascular wall at the aortic root and narrowing of the lumen were taken as the marker of the animal model establishment.The established animal model and normal mice were treated with partial carotid artery dissection injury and vascular injury anastomosis at the same time.After anesthesia recovery according to the random time feeding the mice in the standard in all mice after giving the same treatment,three groups,respectively,for eight hours after the operation,postoperative 48 hours and 21 days after carotid artery damage blood vessels as the next step research specimen,all comes out in the general organization fixed fluid specimens,and fixed at the same time corresponding number 24 hours a day.All specimens underwent immunohistochemistry,immunofluorescence and cell proliferation in order to observe changes of morphology and ultrastructure of Telocytes in atherosclerotic vessels.The experimental method of transmission electron microscopy(TEM)was used to search and verify the Telocytes of carotid artery in ApoE-/-mice and the biological characteristics of Telocytes were observed under high power electron microscope.Results:Telocytes are widely distributed in the myocardial tissue and CD34,CD117 and PDGFR three immunohistochemical index expressionare positive,but not all Telocytes and telopodes express positively.CD34 staining positively Telocytes can be found in the myocardial tissue stroma,a number of elongated telopodes,originating from the cell body,are closely related to neighboring cells,and Telocytes can form a a network structure.CD117 and PDGFR staining could only make telopodes positive-expression,indicating that a certain signal protein in telopodes could express two immunobiological indicators.CD28 staining showed negative results in myocardial tissue,indicating that CD28 was negatively expressed for Telocytes.In addition,CD34 immunohistochemical indexes were positively expressed for Telocytes near the central vena cava in liver parenchyma,and CD34 was also expressed for Telocytes distributed in the liver parenchyma along the central vena cava.The hepatic sinus is located between the hepatic plates,with large and irregular cavities,and there are Telocytes in the "desi"space of the liver.The cells here partially express CD117 immunohistochemical indexes,which cannot completely show the cell body shape of Telocytes,but only show the shape and distribution of telopodes.There are also Telocytes between liver cells in liver parenchyma,and telopodes is positively expressed for PDGFR.CD34 immunohistochemical biological staining is positive for Telocytes in renal medulla and renal cortex,and Telocytes are distributed in the glomerular interstitium of renal medulla and between the epithelial cells of proximal and distal convoluted tubules of renal cortex.Two immunohistochemical indexes,CD117 and PDGFR,are negative staining for Telocytes in renal tissues.In the immunofluorescence double staining experiment,the positive expression of CD28/Vimentin was found in the capillary wall of the heart tissue and some cardiomyocytes,but the negative expression of the two indexes was found in Telocyte.In CD34/CD117 staining,CD34 positive Telocytes were obviously visible,and telopodes could also be expressed,but the expression of CD117 staining Telocytes was not clear,and the morphology,structure and distribution of obvious Telocytes could not be distinguished.In CD34/PDGFR staining,the morphological structure and distribution of Telocytes in heart tissues were obviously observed.The cell bodies of Telocytes were partially expressed for PDGFR and completely expressed for CD34.A three-dimensional network formed between the cell bodies of several Telocytes and telopodes in the double staining of CD34/PDGFR existed in the interstitium of the heart.ApoE-/-mice was detected by double immunofluorescence assay CD34/PDGFR staining of central hepatic venules and peripheral cells in liver showed that PDGFR was generally positive for liver parenchymal cells,so the nature of PDGFR expression for Telocytes could not be clearly distinguished.CD34 positive staining was performed by Telocytes and central venous epithelial cells.ApoE-/-mice liver was evaluated by double staining immunofluorescence biomarkers of CD28/Vimentin,CD34/CD117 and CD34/PDGFR.Fluorescence staining of CD28/Vimentin and CD34/CD117 was common in hepatocytes.In the staining of liver blood sinus and surrounding cells in liver tissues,CD34 positive Telocytes can be clearly distinguished,and PDGFR fluorescence staining is positive for most liver parenchymal cells.CD28/Vimentin staining was generally positive for glomerular cells in the renal medulla.CD34 positive Telocytes were found between adjacent glomerular epithelial cells in renal medulla,while CD117 and PDGFR were negative in both glomerular epithelial cells.In the second part of this experiment,we found that after 12 weeks of high-fat diet typical atherosclerotic changes can be seen in the wall of the aorta in ApoE-/-mice.In carotid artery injury repair in ApoE-/-mice at different stages,Telocytes appeared different biological changes:in the early vascular injury Telocytes begin to gather the breakup of the pipe wall,the number obviously than normal blood vessels inside the membrane cell number;in the middle of the repair of Telocytes sharply reduced the number of phenomena,and Telocytes participate in the vessel wall repair process,completely in the vessel wall after repair.In the control experiment of normal mice,it can be seen that the morphological structure of Telocytes is normal,and the number of them changes in the different stages of vascular wall damage repair,and the time of vascular repair is obviously longer than ApoE-/-mice.The ultrastructure of Telocytes can be clearly observed by transmission electron microscopy.Telocytes are composed of a cell body and telopodes.The cell body shapes are various,oval,triangular and irregular,and the number of telopode is also different.The number of telopode observed in this experiment is 1-5.In atherosclerosis,the ultrastructure of Telocytes in the vascular wall occur to change,the nuclear morphology was irregular,the nuclear membrane became fuzzy,the volume decreased,the internal heterochromatin began to appear,and the nucleolus morphology was irregular.Mitochondria in Telocyte cytoplasm appear vacuolar degeneration,the number is reduced,the distribution is not even,and the Mitochondrial ridge is disappeared.The quantity of rough endoplasmic reticulum decreased.Liposomes of different sizes appear in the cytoplasm.The continuous terminal of the telopode,thickened in diameter,tortuous and swollen,with unclear internal organelles.There are a lot of collagen and elastic fibers around them.Conclusion:In ApoE-/-mice,there are different biological immunologic markers of Telocytes in mouse heart,kidney and liver tissues.Telocytes and telopodes can express different biological immune markers.The immunologic markers expressed by Telocytes are distinct in different tissues and organs.The immunological markers expressed Telocytes in different parts of the same tissue are also different.After 12 weeks of high-fat diet,typical atherosclerotic lipid plaques could be formed in the wall of aorta in the ApoE-/-mice.The Telocytes were present in the outer and middle layers of the arterial wall,and were positively expressed for CD34,CD117 and PDGFR,and negatively expressed for CD28 and Vimentin.Positive expression of telopode for PDGFE and CD34 immune markers.In the repair process of arterial injury in mice,Telocyte is involved in the whole process of tissue repair.The morphologicalstructure and ultrastructure of Telocyte’s organelles in atherosclerotic blood vessels are changed,indicating that Telocytes are involved in the pathogenesis and development of atherosclerosis,and the damage of blood vessel wall is bound to cause the change of Telocytes.In normal arterial vessels,Telocyte’s organelles are clearly visible,the nuclear structure is normal,and the cytoplasm of telopodes are interlinked.更多还原