Isolation And Characterization Of GPC3Positive Subpopulation Of Hepatic Progenitor/Oval Cells In Sprague-Dawley Rats

Objective:To isolate and identify Glypican-3(GPC3) positive subpopulation of hepatic oval cells (HOCs) in an established2AAF/PH Sprague-Dawley (SD) rat model and to further investigate the role of GPC3and hepatic progenitor/oval cells in the tumorigenesis of hepatocellular carcinoma.Methods:Forty-two male SD rats weighed from150g to180g were randomly divided into control group and model group.In order to induce HOCs proliferation, all rats in model group were submitted to4-day2-acetylaminofluorene (2-AAF15mg/kg) daily gavage followed by a two-thirds partial hepatectomy (PH) and then continued daily administration of2-AAF for one week. The rats in both groups were sacrificed and eviscerated the livers for further study at days of0,4,7,9,11,13and16post-PH. The liver tissue was fixed with10%neutral formalin, embedded with paraffin and cut consecutively into5μm-thick sections. Hematoxylin-eosin (HE) stains and immunohistochemical stains using OV-6antibody to detect HOCs were performed. The numbers of OV-6positive cells were counted in10random portal areas. The data was analyzed by one-way ANOVA with SPSS13.0software to determine the peak time of HOCs proliferation.HOCs were assorted by two-step enzyme digestion combined with Percoll density gradient centrifugation at the peak proliferating time. The assorted HOCs were identified by immunohistochemistry and immunocytochemistry using anti-OV-6, CK19and GPC3antibodies. Finally, GPC3positive subpopulation of HOCs was further purified by indirect magnetic activated cell sorting technology.Result:The model using15mg/kg2-AAF followed by a partial hepatectomy could induce HOC proliferation efficiently. The proliferating HOCs were predominantly identified in the portal areas among interlobular bile ducts. No OV-6positive cells were detected in control group and model group of at day0post-PH as well, but variable numbers of OV-6positive cells could be detected in the other different model groups. The numbers of OV-6positive cells were gradually increasing with time until they reached the peak on day9-11post-PH, and then gradually decreasing. HOCs expressing OV-6, CK19and GPC3could be successfully isolated by two-step enzymatic digestion combined with Percoll density gradient centrifugation. Finally, purified GPC3positive subpopulation of HOCs was obtained by indirect magnetic activated cell sorting technology.Conclusions:1. The model using15mg/kg2-AAF followed by a partial hepatectomy can induce HOC proliferation efficiently. The peak time of HOC proliferation occurs on day9-11post-PH.2. The isolated HOCs express OV-6, CK19and GPC3. This provides further evidence that GPC3is a new cell marker for HOCs.3. Indirect magnetic activated cell sorting technology can be an efficient method to separate and purify GPC3positive subpopulation of HOCs.