Observe The Biological Characteristics Of Hepatic Oval Cells And Explore The Changes Of TGF?1/ALK1 Signaling Pathway In Inflammatory/non-inflammatory Liver Injury-repair Model

Objective:One:To explore the liver micro-environment affect the fate of the hepatic oval cells?HOCs?,observe the cytologic features of HOCs in non-inflammatory proliferation environment and in inflammatory proliferation environment by non-inflammatory liver injury-repair based on HOCs proliferation model and inflammatory liver injury-repair based on HOCs proliferation model.Two:To explore the expression of TGF?1,ALK1,Smad5,Id1,and the number of OV6-positive HOCs at initial-stage of injury-repair,mid-stage of injury-repair,late-stage of injury-repair in non-inflammatory liver injury-repair based on hepatocytes proliferation model,non-inflammatory liver injury-repair based on HOCs proliferation model and inflammatory liver injury-repair model,detected the changes of TGF?1,ALK1,Smad5,Id1 in different liver injury-repair model and different point,analyzed relationship between the proliferation of HOCs and the changes of TGF?1/ALK1 signaling elements.Methods:One:Fifteen Sprague Dawley rats were randomly divided into three groups:control,non-inflammatory liver injury-repair based on HOCs proliferation model,inflammatory liver injury-repair based on HOCs proliferation model.2-acetaminofluorenced?2-AAF?combined two thirds partial hepatectomy protocol was established as non-inflammatory liver injury-repair based on HOCs proliferation model.2-AAF combined carbon tetrachloride?CCl₄?protocol was established as inflammatory liver injury-repair based on HOCs proliferation model.Liver histological changes were observed by hematoxylin and eosin staining.The expression of EpCAM,CD133 and OV6 of HOCs was detected by immunohistochemistry,average optical density values were measured.The ultrastructure changes of HOCs were observed by transmission electron microscopy.Two:Sixty Sprague Dawley rats were randomly divided into four groups:control group,one third partial hepatectomy was established as non-inflammatory liver injury-repair based on hepatocytes proliferation model,2-AAF combined one third partial hepatectomy protocol was established as non-inflammatory liver injury-repair based on HOCs proliferation model,subcutaneous injection CCl₄ was established as inflammatory liver injury-repair model.Liver tissue structure changes was observed by HE staining.The localization of TGF?1,ALK1,Smad5 and Id1 were observed by immunohistochemistry,and hepatic oval cells were identified via OV6 by immunohistochemistry.The expression level of TGF?1,ALK1,Smad5 and Id1 were detected by Western Blot.Results:One:We successfully established non-inflammatory liver injury-repair based on HOCs proliferation model and inflammatory liver injury-repair based on HOCs proliferation model.Hematoxylin and eosin staining:The liver structure of control group rats'liver were normal;non-inflammatory liver injury-repair model rats'liver were absent with inflammatory cells infiltration;inflammatory liver injury-repair model rats'liver were fibrosis formation period with large number of inflammatory cells infiltration.Immunohistochemical:EpCAM-positive,CD133-positive,and OV6-positive HOCs were found both in non-inflammatory and inflammatory liver injury-repair model,the expression level of EpCAM?CD133?OV6 of inflammatory liver injury-repair model were significantly higher than non-inflammatory liver injury-repair model?P<0.05?.Transmission electron microscopy:type I,II,and III of HOCs were found both in non-inflammatory and inflammatory liver injury-repair model,however,there were more heteromorphic HOCs in inflammatory liver injury-repair model than non-inflammatory liver injury-repair model?P<0.05?.And we found ductular reaction in inflammatory liver injury-repair model.Two:HE staining found hepatocytes with bi-nuclear dividing phase,suggested that we successfully established non-inflammatory liver injury-repair based on hepatocytes proliferation model.OV6-positive HOCs were found in non-inflammatory liver injury-repair based on HOCs proliferation model and inflammatory liver injury-repair model suggested that we successfully established non-inflammatory liver injury-repair based on HOCs proliferation model and inflammatory liver injury-repair model.TGF?1,ALK1,Smad5 and Id1 expressed in all of the models detected by immunohistochemistry.Western Blot:expression of TGF?1 in non-inflammatory liver injury-repair based on hepatocytes proliferation model was gradually increased,expression of TGF?1 in non-inflammatory liver injury-repair based on HOCs proliferation model was firstly increased and then decreased,the ALK1 expression trend were consistent with the corresponding model of TGF?1 trend.Expression of Smad5 in non-inflammatory liver injury-repair based on hepatocytes proliferation model was firstly decreased and then increased,expression of Smad5 in non-inflammatory liver injury-repair based on HOCs proliferation model was gradually decreased.Expression of Id1 in non-inflammatory liver injury-repair based on hepatocytes proliferation model and non-inflammatory liver injury-repair based on HOCs proliferation model was gradually increased.In inflammatory liver injury-repair model,the expression of TGF?1,ALK1,Smad5 and Id1 were gradually increased as liver injure aggravating,while during the recovery stage when stopped the injection of CCl₄ the expression of TGF?1,ALK1 and Smad5 were decreased,but the expression of Id1continuously increased.The number of OV6-positive HOCs peaked at first week in the non-inflammatory liver injury-repair based on hepatocytes proliferation model and non-inflammatory liver injury-repair based on HOCs proliferation model,peaked at sixth week in the inflammatory liver injury-repair model.Conclusions:One:2-AAF combine two thirds partial hepatectomy protocol can successfully establish non-inflammatory liver injury-repair based on HOCs proliferation model,2-AAF combine CCl₄ protocol can successfully establish inflammatory liver injury-repair based on HOCs proliferation model,one third partial hepatectomy can successfully establish non-inflammatory liver injury-repair based on hepatocytes proliferation model,subcutaneous injection CCl₄ can successfully establish inflammatory liver injury-repair model.Two:HOCs in inflammatory environment possessed stronger tumorgenicity than HOCs in non-inflammatory environment.Subcellular structure of HOCs may change at early stage in inflammatory environment.Three:TGF?1/ALK1/Smad5/Id1 signaling pathway elements dynamic expression are different in non-inflammatory and inflammatory liver injury-repair model,different in HOCs proliferation based and hepatocytes proliferation based.