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(Hepatic Stellate Cells) in Diagnosis of Liver Fibrosis

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(Hepatic Stellate Cells) in Diagnosis of Liver Fibrosis Gastroenterology & Hepatology: Open Access Research Article Open Access Ito stellate cells (hepatic stellate cells) in diagnosis of liver fibrosis Introduction Volume 10 Issue 4 - 2019 Adverse outcome of most chronic diffuse liver lesions of various etiologies including chronic hepatitis C (CHC) is liver fibrosis in the Vladimir Tsyrkunov,1 Viktor Andreev,2 Rimma development of which main participants are activated fibroblasts. Kravchuk,3 Iryna Kаndratovich1 Activated hepatic stellate cells (HSC) are the main source of activated 1 1,2 Department of Infectious Diseases, Grodno State Medical fibroblasts. University, Belarus 2 HSC, a synonym - stellate cells of liver, Ito cells, perisinusoidal Department of Histology, Grodno State Medical University, Belarus lipocytes, stellate cells. HSC were firstly described in 1876 and named 3Department of Science, Grodno State Medical University, by K. Kupffer stellate cells T. Ito, finding in them a drop of fat firstly Belarus marked them as lipophagic («shibo-sesshusaibo») but then finding that the fat elaborated by the cells from glycogen named them as Correspondence: Iryna Kаndratovich, Department of fat-storing cells or Ito sells («shibo-chozosaibo»).3 In 1971, K.Wake Infectious Diseases, Grodno State Medical University, Gorkogo str 80, Grodno 230009, Belarus, Tel +375152434286, Fax proved the identity of stellate cells of Kupffer and Ito cells and that +375152434286, Email these cells are “stored” vitamin A.4 Received: October 29, 2018 | Published: July 25, 2019 About 80% of vitamin A which is in the body accumulates in the liver and up to 80% of liver retinoids deposited in fat droplets of HSC. Ethers of retinol in formulation of chylomicrons entered to Methods hepatocytes, where they are converted to retinol and form of vitamin A complex with retinol binding protein (RBP), which is secreted in Lifetime liver biopsy obtained by performing aspiration biopsy perisinusoidal space where it deposited by cells.5 of the liver of patients with CHC (HCV ribonucleic acid+). Patients gave theirs written informed consent. For light microscopy semifine Established by K. Popper close relationship of HSC with the sections of liver biopsy samples of patients 0,5х2mm in size were hepatic fibrosis demonstrated theirs not static, but dynamic function. fixed by double fixation method: firstly by the Sato Taizan technique.8 It means the ability to be directly involved in the remodeling Then tissue samples for 1 hour additionaly fixed in 1% osmium of intralobular perihepatocellular matrix.6 The main method of fixative prepared with 0.1M phosphate buffer of Zerensen, pH 7.4. morphological study of liver conducted by assessing the changes For better identification of intracellular structures and interstitial in lifetime biopsy materials is light microscopy. This method in substance on semifine sections in 1% osmium tetroxide was added clinical practice allows establishing the stage of inflammation activity potassium dichromate (K2Sr2O7) or crystals of chromic anhydride and stage of chronization.7 The disadvantage of this method is low (1mg/ml). After dehydration of samples in series of alcoholic resolution which does not allow evaluating features of cell structure, solutions of increasing concentrations and acetone they were intracellular organelles, insertions, functional characteristics. Lifetime placed in the prepolymerized mixture of butyl methacrylate and electron-microscopic study of ultrastructural changes in the liver styrole and polymerizing at 55°C. Semifine sections (thickness of makes it possible to supplement the light microscopy data and raise 1 microeter) were sequentially stained with azure II-basic fuchsine. their diagnostic value. Microphotographies obtained with using of digital video camera In this regard the identification of the HSC, study of their (Leisa FC 320, Germany). phenotype during transdifferentiation and determining the intensity Electron-microscopic study was performed in samples of liver of their proliferation are the major contribution to the prediction biopsy materials 0,5х1,0mm in size fixed with 1% osmium tetroxide of outcomes of liver disease as well as in the patomorphology and on 0.1M buffer of Millonig, pH 7.4, at +4°C for 2 hours.9 After pathophysiology of fibrogenesis. The aim of research is to introduce dehydration in alcohols of increasing concentrations and acetone structural and functional description of HSC on the results of samples were embedded in Araldite.10,11 From these blocks on cytological identification of lifetime liver biopsies. ultramicrotome Leica EM VC7 (Germany) were prepared semifine sections (400 nm) and stained with methylene blue. Specimens Abbreviations: CD, cluster of differentiation; CHC, chronic viewed under the light microscope and the same type of site chosen hepatitis C; HCV, hepatitis C virus; HSC, hepatic stellate cells; ICAM, for further study of ultrastructural changes. Ultrathin sections (35 nm) intercellular adhesion molecule; MMPs, matrix metaloproteinases; contrasted with 2% solution of uranyl acetate per 50% of methanol12 PDGF, platelet-derived growth factor; RBP, retinol-binding protein; and lead citrate by E.S. Reynolds.13 Electron-microscopic spesimens RER, rough endoplasmic reticulum; TIMPs, tissue inhibitors of were studied in the electron microscope JEM-1011 (JEOL, Japan) matrix metaloproteinases with an increase of 10000–60000 at an accelerating voltage of 80 kW. Submit Manuscript | http://medcraveonline.com Gastroenterol Hepatol Open Access. 2019;10(4):213‒219. 213 © 2019 Tsyrkunov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Ito stellate cells (hepatic stellate cells) in diagnosis of liver fibrosis ©2019 Tsyrkunov et al. 214 For images was used a set of digital camera Olympus MegaViewIII The amount of lipid droplets in the inactive HSC is 30 or more. (Germany) and programs for processing of images iTEM (Olympus, They are similar in size and are adjacent to each other, pressing Germany). into the nucleus and pushing it aside periphery (Figure 2). Small Results and discussion inclusions can be placed between large droplets. The color of droplets depends on the fixator and staining of material. In one case, they are HSC located in perisinusoidal space (Disse) in recesses between bright (Figure 2A), in the other they are dark green (Figure 2B). hepatocytes and endotheliocytes and have long processes that penetrate deep between hepatocytes. In the majority of publications At electron microscopy on a background of the light lipidic 22 devoted to this population of HSC is a schematic representation of substrate more osmiophil marginal ring is formed (Figure 2C). Most them which allows only indication of “territorial” belonging of HSC of the “inactive” HSC along with large lipid inclusions small amount in the liver and in relation to others of their “neighbors” (Figure 1). of cytoplasmic matrix poor on mitochondria and rough endoplasmic reticulum (RER) is seen. However, compartments of moderately developed Golgi complex in the form of stacks of 3-4 flattened cisterns with slightly extended ends are clearly seen (Figure 2S). Figure 1 Diagram of HSC location in perisinusoidal space of Disse, an internete resource. HSC have close contact with endotheliocytes through the incomplete basement membrane components and interstitial collagen fibers. Nerve endings penetrate between HSC and parenchymatous cells, wherefore the space of Disse defines as the space between the plates of parenchymatous cells and HSC complex and endotheliocytes.14 It is supposed that HSC become from poorly differentiated 15 mesenchymal cells transverse septum of developing liver. The Figure 2 HSC which are in inactive condition. experiment revealed that in the formation of HSC hematopoietic stem cells participates, and that this process is not caused by cell fusion.16 (A) Hepatic stellate cells (HSC) of round shape with a high content of lipid droplets with a light color (white arrows), hepatocytes (Hep) with Sinusoidal cells, first of all HSC, play a leading role in all forms the devastated cytoplasm (black arrow); B, HSC with lipid droplets of dark of liver regeneration. Fibrotic liver regeneration occurs as a result of color (white arrows), in close contact with macrophage (M); A, B, Semifine the inhibition of HSC stem functions and stem cells of marrow.17 In sections, stained with azure II - basic fuchsine, microphotography, zoom 1000; human liver HSC constitute 5-15% being one of 4 types of sinusoidal C, HSC with plenty of lipid droplets (more then 30) having an irregular shape, cells with mesenchymal origin: Kupffer cells, endotheliocytes and zoom 6000 (Hep – hepatocyte); D, Ultrastructural components of HSC: lipid Pit-cells. Within pool of sinusoidal cells 20-25% of leukocytes are droplets (L), mitochondria (orange arrow), rough endoplasmic reticulum (green arrows), the Golgi complex (red arrow), zoom 15000; (C-D) Electron detected.18,19 diffraction patterns. There are lipid inclusions with retinol, triglycerides, phospholipids, Under certain conditions activating HSC obtain mixed or cholesterol, free fatty acids, a-actin and desmin in the cytoplasm transitional phenotype combining morphological characteristics of of HSC.20 To visualize HSC auric chloride staining is used. The lipid like and fibroblast-like cells Figure( 3).22 experiment found that differentiation marker of HSC from
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