The Research Ofultrastructure Of Hepatic Oval Cell And Sequential Changesin Biliary Obstruction Rat

Background and ObjectiveIn the liver and gallbladder surgery clinical, biliary obstruction and cholestasis are common pathological states. Longer obstructioncan cause permanent liver damage. The clinical treatment is ineffective, related to the pathogenesis of basic research has practical significance.The liver progenitor cells,oval cells, has been found in the chronic liver disease such as cirrhosis of the liver, sub-massive hepatic necrosis of liver regeneration, children with congenital extrahepatic biliary atresia, hereditary hemochromatosis, chronic viral hepatitis, hepatocellular carcinoma. This hepatic progenitor cells located in periportal areas, fiber spacing, periportal zone adjacent hepatic parenchyma, and inflammatorypseudo-lobular borders. With the specific markers of the oval cells found, research about oval cells has been hot, especially in the researchof liver damage repairing and liver fibrosis. Other studies have provedthat multiple organ fibrosis is accompanied bythe reactivation of the gene related embryonic development and kinds of cytokines.Theseprovide the latest way in the research of hepatic injury protection, treatment and the formation mechanism of liver fibrosis.In this study, we explore the roles of oval cells and cytokines related to the pathological process in the process of biliary obstruction and hepatic fibrosis, using histological and PCR techniques, by ligating Wistar rats’common bile duct to replicatethe pathological states of biliary obstruction.Methods:Male Wistar rat60.Body weight250-300g, health and no obvious deformity. They were randomly divided into experimental group and control group. One group (30only) as the experimental groupis carried out nearly portal single-lane ligation (BDL); another group (30only) as control group is carried outonly laparotomy but not ligation. Postoperative raised under the same conditions.The1,2,3,7,14,21day after the operation, five in each group were sacrificed. Take right lobe of liver4%paraformaldehyde fixed histological analysis. Take liver left lobe-80℃frozen for RT-PCR detection reserved, After liver middle small slices of liver tissue, fixed reserved for electron microscope. 1Through the naked eye, and HE staining of bile duct obstruction inliver morphological changes at different time points.2Through immunohistochemistry, observe the number and distribution of oval cells at different time points, by OV-6marker。3Through the projection electron microscope to observe the ultrastructure of oogonium changes4Through the PCR detection to every purpose:Hepatocyte growth factor (HGF), Collagen I (COL I), observe each gene expression at different time points.Results1-. General results:After Wistar ratbiliary obstruction,the liver is yellowing and swelling, oedema and congestion. We can find Formalin fixed part of rat liver with the rough surface samples andgetting grey, the performance of early liver fibrosis. The control group: liver specimens, each lobe rosy and smooth surface.2> HE dyeing results:We can see little neutrophil and lymphocytes at the first day; we cana few new born little bile duct without the lumen, a few neutrophil and lymphocytes at the second day;we can see new born of tiny bile ducts and without the lumen small bile duct, neutrophil and lymphocytes more than the second day significantly, liver cell mild degeneration, visible multifocal and flake necrosis, liver tissue collect abbacy visible bile duct proliferation and fibrous tissue cell hyperplasia and an occasional bile duct dilation at the third day; at the seventh day liver cells appear degeneration necrosis incidence increased worse progressively, and degree of hemorrhage and necrosis seen flake, liver tissue bile duct proliferation and fiber hepatis visible; at14th days lobular structure damage, visible liver cell vacuoles degeneration and necrosis; Postoperative21days lobular structural damage more serious, a lot of necrosis of liver cells. Over time and liver damage, fibrosis degree progress gradually deteriorated.3、Immunohistochemical results:OV-6immunohistochemical staining cell number and positive distribution with obstruction the extension of time and increase, experimental group and control group have obvious difference. OV-6positive cells express parts mainly in collect abbacy bile ducts and liver interstitial new born bile duct and no small bile duct cells lumen of cytoplasm.4、Electron microscope observationsresultsControl group:normal liver tissue and liver cell structure.BDL group:postoperative1day a small perivascular ovoid nuclear cells, a small round blunt cytoplasmic projections, nucleoplasm ratio is big, the quality is less and less number of organelles;2days between liver cells can also find some ovoid cell, cell aspect or bell-shaped fat marquis, kernel approximation ovoid, nuclear, nuclear pulp than the big, less cytoplasm, poor development;3days after partial liver blood sinus endothelial injury, fracture collapse endothelial cells, endothelial cell nucleus is small, heterochromatin is thick dyeing of briquette, edge set under nuclear membrane.Kupffer cell to gobble up the bubble number is more, see more ovoid nuclei in intercellular space;7days of postoperative liver cell plate appear larger gap, or only remaining cells framework, some liver cells are irreversible damage, some cells apoptosis necrosis, visible polymorphonuclear leucocyte exist between liver cells, some cells only can distinguish the size differences of nuclear, cells beyond resolution details, in the form of distraction, still can find a small amount of round to oval nuclei of survival cells, has been in the direction of liver cell differentiation;After14days have some cuboidal cells between liver cells,4-5cells forming block, between cells visible tubular structure, has a close connection between cells, at the ends of the small tube with microvilli, into the lumen contents, the nuclear shape approximation egg circle, but not very rules, have cave, organelles, secondary development of nuclear membrane cell block connected to the liver cells have;21days after some collagen fiber bundle of tip DouZhou clearance position.Liver cells visible ovoid between nuclear cell block, some cells contain a large amount of lipid droplets;Some cells are fibroblasts, the cells around the presence of a large number of collagen fibers;The rest of the cell properties.5、RT-PCR resultsIn the control group and BDL group, we can detect all purpose gene expression in the liver tissue. Statistical analysis showed that the two groups COL I have the statistically significantfrom the first day, HGF have the statistically significantfrom the third day. Between BDL groups of different days, COL I in3th-7th days have no statistically significant, HGF in lth-3th days and7th-21th days have no statistically significant, while the other statistically significant. The purpose to express a growing trend among the genome, COL I grow the fastest at lth-3th days, HGF grow the fastest at3th-7th days.Conclusion1、This study find that the liver of the BDL rats present cholestasis, progressive aggravating fibrosis performance. Oval cells Participate in the damage repair process afterbile duct obstruction. Its timing change is similar with antifibrosis cytokines, so it may participate in anti fibrosis process.2、Research found that the liver fibrosis in the experimental groupsare significantly higher related gene expression than the control groups, with the increase of obstruction of the amount of days express significantly increased. COL I changes represent fibrosis process, TGF-β1is related to the liver fibrosis process, TGF-α and HGF are related to theantifibrosis process.3、The study found that the period,3th-7th days, is special in the fibrogenic process,3th day Beforethe3th day the main performance is the promoting fibrosis process; from the3th dayanti fibrosis performance begin to appear, promoting fibrosis trend abate, fibrosis become slowly; afterthe7th day the liver fibrosis is the continuous progress.