| Objective: Pericytes are distributed in the capillaries of wall in the whole body.They are microvascular cells surrounding the endothelial cells.They have some systolic function and regulate the blood flow by changing the diameter of the blood vessels.Together with the endothelial cells,they form Blood-brain barrier,Have a certain defense function from outside.In addition,Pericytes also have a multidirectional differentiation potential that can differentiate into many different cell types.Mesenchymal stem cells are a population of cells that are first isolated from the stromal portion of many connective tissues that can differentiate into various mesodermal lineages and aroused general interest in studies about adult mesodermal progenitor cells.Pericytes are derived from the mesoderm,a primitive cell of mesenchymal stem cells and have strong multiple differentiation potential.Pericytes exist in skeletal muscle as perivascular mesenchymal precursor cells,where they contribute to the regeneration of muscle fibers.Pericytes derived from brain tissue,retina,liver tissue,adipose tissue and skeletal muscle tissue have now been demonstrated to be pluripotent stem cells with the multi-directional ability to differentiate.There are many common properties between pericytes and mesenchymal stem cells,under some conditions,pericytes can differentiate into adipocyte lines,fibroblast lines,odontoblasts,osteoblastic lines,cartilage cell lines.According to its potential characteristics of multi-directional differentiation,pericytes can participate in the repair of various tissues and organs.Pericytes can differentiate into osteocytes,osteoblasts,chondrocytes and adipocytes,and participate in vascularization.The summary reveals the role of vascular pericytes in the differentiation of vascular smooth muscle and in the reconstruction of neovascularization.Methods: 1.Take 3 healthy 3-week-old SD rats weighing 110g-130 g,under anesthesia take the heart,cut the ventricle into 3 × 3mm pieces of tissue,then add the neutral protease II and collagenase B And highly purified bovine serum albumin mixed to make digestive enzymes.After centrifugation and resuspension,the primary cultured pericytes of cardio-vascular cells were added to the culture medium with the primary cultured pericytes in proportion,and the cell culture was passaged to the third generation,as P3;2.Immunofluorescence qualitative analysis and cell flow cytometry were used to identify the extracted primary pericytes;3.A certain amount of TGF-β was added to the pericytes medium,the high-glucose DMEM medium and the mesenchymal stem cell F12 medium as differentiation medium to differentiate cultivate for 21 days;4.Morphological changes of pericytes before and after directional differentiation of smooth muscle cells were observed by inverted phase contrast microscope;5.The changes of SMA,SM22-α,PDGFR-β,CD146-related pericyte proteins marker were analyzed by immunofluorescence before and after differentiation,including single and double immunofluorescence staining;6.Western Blot Western blot was used to quantitatively analyze the changes of SMA,SM22-α,CD146 and PDGFR-β-related protein markers before and after differentiation;7.Pericytes independent matrigel angiogenesis experiments were used to assess the ability of angiogenesis;8.Transwell chamber migration experiments were used to assess the migration of cardiac pericytes during angiogenesis.Results: 1.Observation of Cell Morphology Before and After Differentiation and Cultivation: The primary cells are cultured for 24 hours after extraction and can be seen in an inverted phase contrast microscope with star-shaped or polygonal cells,with many long protrusions per cell protruding and adherently growing,with no contact Inhibition of growth;Using differentiation medium for 21 days of differentiation culture,observed under the microscope,the cell morphology of irregular,multiple protrusions polygons become regular,reduce or even disappear fusiform.2.Identification of primary pericytes:strong positive expression of PDGFR-β,NG2,CD146 and other related pericytes markers by flow cytometry,immunofluorescence and Western Blot Western blot.3.Immunofluorescence staining results before and after differentiation:Immunofluorescence staining of cell slide showed that pericytes showed strong positive expression of PDGFR-β,CD146 and other related markers before differentiation and differentiation.PDGFR-β,CD146 The expression of SM22-α and SMA-associated smooth muscle cell marker protein in differentiated cells was increased compared with that before differentiation.4.Western Blot Western blot results before and after differentiation into smooth muscle cells: The results showed that the expression of CD146,PDGFR-β and other related pericytes markedly decreased compared with that of undifferentiated culture after differentiated culture.In contrast,differentiation culture After immunofluorescence staining SM22-α,SMA and related smooth muscle cell marker protein expression enhanced compared to before differentiation: to further illustrate the perivascular differentiation of vascular smooth muscle cells to vascular smooth muscle cells.5.Pericytes were cultured on Matrigel for 8 hours.After 16 hours,a large number matured vascular structures were formed.As time passed,the vascular structures became stable and connected into a network with clear and regular branches.After 24 hours,there were A small part of the vascular structure began to decompose;48hours later,many vascular structures began to decompose,vascular network connection structure disappeared.6.Transwell chamber migration experiments show that pericytes have a certain ability to migrate,and 24 hours when the cell migration was significantly more than 12 hours of cell migration.Conclusions: The experiment proves in some conditions,pericytes have ability of differentiation into smooth muscle cells,migration and blood vessels formation independently. |
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