| 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. |
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