Study Of The Effect Of Hepatic Stem Cells In The Liver Regeneration After Injury And To The Hepatic Carcinoma Cells

That the relative roles of intrahepatic/extrahepatic stem cells and hepatocytes in liver regeneration in adult was still one core question urgently to answer in the liver regeneration field. And we had ever proposed liver valley hypothesis to elucidate it.Histiocytopathology(big hepatocyte karyomitoses,small karyomitoses and small bile ducts/Hering's canal/oval cells proliferation and so on),frozen section green fluorescence and immunohistochemistry in situ were employed to dynamicly observe:the mouse survivals after cross-sex GFP+ bone marrow transplantation which were irradiated before transplantation, or cross-sex GFP+ gonad transplantation,were administrated without other treatment,with the sham operation,with monocrotaline to inhibit hepatocytes division and growth,with 2/3 partial hepatectomy repeatly,with monocrotaline and 2/3 partial hepatectomy repeatly,with CCl4 injury after injection of monocrotaline to inhibit hepatocytes division and growth or not.1.1 The mice were divided to the control group after cross-sex bone marrow transplantation that the bone marrow of GFP+ C57 male mice were transplanted into ordinary C57 female mice irradiated with lethal dose before transplanted.After survial for six weeks,we found that karyocytes in the peripheral blood had green fluorescene on the whole and mononuclearcells in the hepatic sinus had green fluorescene mainly.A few small cells around the small bile duct in the portal area had green fluorescene.And only few hepatocytes in the hepatic plate had green fluorescene by chance. These might support literatures that hepatic stem cells origing from bone marrow could participate in the physiological renewal of hepatocytes in very small ratio without obvious liver injury.The results from the sham operation group that was opened the abdominal cavity ,not given PH and closed the abdominal cavity were similar with those of the control group.1.2 The mice were given cross-sex bone marrow transplantation and monocrotaline which inhibited mature hepatocytes division and growth.Hepatocytes in the hepatic plate with green fluorescene were obviously more than those of the control group.Monocrotaline could inhibit division and growth of hepatocytes and the physiological renewal of hepatocytes was fulfilled by proliferation and differentiation of extrahepatic/intrahepatic stem cells in the portal area at this time.And we could find that a few small cells including the bile duct in or around the portal area with green fluorescence.And a few small cells and hepatocytes in the hepatic plate indirect approximation with the portal area and in the hepatic plate near the central veins were also with green fluorescence.These hinted that bone marrow (extrahepatic) stem cells origining from the blood circulation could replenish intrahepatic stem cells near the portal area,and some could go through the hepatic plate from the hepatic sinusoid bloodstream pathway from perilobule to the central vein and could proliferate and differentiate to mature hepatocytes.1.3 The mice were given PH many times regularly after cross-sex bone marrow transplantation and there were a lot of big karyomitosis with liver regeneration.But contrasting with the control group,there were a little proliferation of the small bile duct/COH and a few small karyomitosis besides the hepatic sinus by change.Our results hinted that not only intrahepatic stem cells but also extrahepatic stem cells participated in the liver regeneration in some degree,notwithstanding liver regeneration were accomplished by mature hepatocytes mainly when the capability of division and hyperplasia of mature hepatocytes was not inhibited.And intrahepatic stem cells could be recruited by extrahepatic stem cells.1.4 The mice were given monocrotaline which inhibited division and growth of hepatocytes after cross-sex bone marrow transplantation and given PH many times regularly.There were more obvious proliferation of the small bile duct/COH and OCs.We could nearly not find the big karyomotisis and the small karyomotisis were obviously more.There were more hepatocytes in the hepatic plate,the small bile duct/COH and OCs with green fluorescent than those of the control group.These observation hinted that OCs could origin from the resident small bile duct/COH without green fluorescence and from the small bile duct/COH and small cells nearby from bone marrow with green fluorescence.Cytokeratin 19 is positive in the small bile ducts with proliferation in immunohistochemisty.AFP is negative or weak positive in the oval cells,obviously positive in the bigger cells which had already changed to the small hepatocytes,and negative in the hepatocytes.1.5 The mice were given CCl4 after cross-sex bone marrow transplantation to induce hepatic fibrosis and contrasted with the control group.The cells with green fluorescence were more and majority were infiltration of the single nucleus small cells with degeneration and injury of hepatocytes.Hepatocytes with green fluorescence were more.There were more OCs regeneration around a few portal area.There were some big karyomitosis but obviously few than those of the PH group without monocrotaline which inhibited division and growth of hepatocytes.And there were obviously more small karyomitosis.These hinted that there were more extrahepatic/intrahepatic stem cells participating in the liver regeneration than those of the PH group,using CCl4 with more widespread degeneration of mature hepatocytes and even focus necrosis and the capability of division and regeneration of hepatocytes not more than that of the PH group.1.6 If we used monocrotaline which inhibited division and growth of hepatocytes at the same time,the proliferation reaction in the juncture of the lobule and the small bile duct was obvious.Some proliferation focus of the small bile duct/OC had not green fluorescence and some had.There were more hepatocytes with green fluorescence.The inflammatory reaction cells and some small cells near the portal area could have green fluorescence.Extrahepatic/intrahepatic stem cells could effectivly participate in liver regeneration with CCl4.And extrahepatic stem cells could replenish the small bile duct which were the position of intrahepatic stem cells.If we stoped CCl4,hepatic fibrosis could relieve step by step.If we gave these mice the GFP+ bone marrow cells in tail vein,hepatic fibrosis could also relieve step by step,which was similar with the correlated reports before.2. There were stem cell pool hypothesis including gonad stem cell pool hypothesis,raising about the same time with liver valley hypothesis.And we took research with GFP+ gonad transplantation.And we found that there were nearly not mononuclearcells with green fluorescent in the peripheral blood smear in mice with PH or CCl4 after gonad transplantation.If we took monocrotaline which inhibited hepatocyte division and growth,there were conspicuous regeneration of the small bile duct/COH and OCs and we could find very few mononuclearcells or hepatocytes with green fluorescent in the liver plate of the whole liver tissue section in a few mice.These hinted that gonad stem cells did nearly not play a part in the physiological renewal of hepatocytes or liver regeneration after pathological injury.The liver valley hypothesis was preliminary verificated based on the our experiment that both intrahepatic/extrahepatic stem cells and hepatocytes contributed to the liver regeneration,in which hepatic stem cells play a more impportant role when the hepatocytes were injured and their regeneration capacity decreased, especially the stem cells from bone marrow considering that extrahepatic stem cells could fill up intrahepatic liver stem cells.The Second PartWe entrusted other person to breed our mice,and he made a mistake to give our mice after bone marrow transplantation with lethal irradiation N-nitrosodiethylamine(DEN) in our first part experiment.And we found tumor in mice after 18 weeks,and there were some the small cells(cells from bone marrow) with the green fluorescence in the borderline of the tumor and the normal tissue.Based on this discovery by chance,we thought what role the stem cells were around the tumor?In order to get the answer,we did the second part experiment.We cultured the CBRH-7919 hepatic carcinoma cells and WB-344 hepatic stem cells of rat together,and we found that WB-344 rat hepatic stem cells promoted apoptosis of CBRH-7919 rat hepatic carcinoma cell and inhibited it grew with the TGF-?/Smad way in the microenvironment of the hepatic cancer.And this result gave new theory base to treat the hepatic cancer patients.The specific and concise results were as followed.The result from flow cytometer indicated that the apoptosis rate of CBRH-7919 rat hepatic carcinoma cell increased apparently with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The soft agar clone formation experiment indicated that with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system,the amount of the CBRH-7919 rat hepatic carcinoma cells clones decreased apparently.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The four transwell cell infestation experiment indicated that the infestation ability were all negative.This result indicated WB-344 rat hepatic stem cells did not affect the infestation ability of CBRH-7919 rat hepatic carcinoma cells.Demi-quantitation RT-PCR analysis displayed that PTEN was upregulation and bone morphogenetic protein 4 (BMP4), Bcl-2, and c-Myc was downregulation in CBRH-7919 rat hepatic carcinoma cells with the increment proportion of the WB-F344 rat hepatic stem cells in the co-culture system.This result indicated that WB-344 hepatic stem cell could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The result from Western blot hybridization analysis indicated that with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system,the expression level of Smad 4 and TGF-?RII increased.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells regulating the TGF-?/Smad way to promote the apoptosis of CBRH-7919 rat hepatic carcinoma cells.In the thirtieth days,in the mice with the tumor with the increment proportion of WB-344 rat hepatic stem cells in the co-culture system,the volume of tumor decreased,life span increased conspicuously and Kaplan Meier survival analysis had statistics significance.This results indicated that WB-F344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells in nude mice in vivo.Contrasting with the experiment groups,the malignancy grade of tumor increased,the ration of nucleus to cytoplasm increased,there were some mononuclear or polynuclear cells in many area in the conrol group,and the heteromorphism of tumor cells degraded with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system.This results indicated that WB-F344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.