The Dedifferentiatin Of Mouse Hepatocytes And Its Mechanism

?Background and Objective?The traditional view of organization development and cellular differentiation is a one-way process,namely stem cells/progenitor cells differentiation to somatic cells.Therefore,under the condition of normal update or tissue damage,stem cells/progenitor cells play an important role in the process of tissue regeneration.So far,however,this important role of stem cells/progenitor cells was only confirmed in one of the few organs,the corresponding research evidence was lacked in most other organs,including the liver.The liver's Stem Cells/Progenitor Cells called Hepatic Progenitor Cells(Hepatic Progenitor Cells,HPCs),it can differentiation to both hepatocytes and bile duct epithelial cells.Due to its expressing many kinds of cell markers,HPCs lack specific markers of its mine,and also its origin so far is not very clear.The traditional view consider HPCs deriving from the Canal of Hering.There is also studies suggest that HPCs may derive from bone marrow stem cells or hepatic stellate cells,etc.In addition,there are also studies have shown that hepatocytes can also be one of another source of HPCs.Due to its special position and function,liver make its become one of the very special organs.As its main substance composition and function unit,hepatocytes therefore have different physiological features from other somatic cells,such as unlimited proliferation and conversion tobiliary epithelial cells,etc.The latest researchs show that hepatocytes can also dedifferentiation to HPCs in vivo and in vitro.All these studies,however,lack neither rigorous linage tracing evidence,nor deeper research about the mechanism of hepatocytes dedifferentiation.Hepatic nucleus factor 4?(HNF4?)is the group of transcription factors enriched in liver and plays an important role in the development of liver and maintaining of mature hepatocytes function.Studies have shown that HNF4? can induce the differentiation of HCC cells into hepatocyte-like cells,but its role in the differentiation and dedifferentiation of mature hepatocytes is not reported yet.In this study,using a variety of transgenic mice and liver injury models,we aimed to clarify the contribution of HPCs in liver regeneration in different conditions and make sure whether hepatocytres can be sources of HPCs and discussing its mechanism primarily.Methods1.The generation of hepatocytes specially expressing green fluorescent protein(GFP) transgenic miceThis research adopts mTomato-mGFP dual fluorescent transgenic mice.Thyroid binding globulin(TBG)is expressed in hepatocytes specifically.Serum type ? adeno-associated virus carrying TBG promoter and introns(AAV8-TBG)can drive the downstream genes specifically expressed in the mature hepatocytes.After tail intravenous mTomato-mGFP mice with AAV8-TBG carrying Cre recombinant enzyme gene(AAV8-TBG-Cre),hepatocytes specifically express GFP and therefore are marked with green fluorescence,other liver cells are marked with red fluorescence.2.Liver regeneration in acute liver injuryTail intravenous mTomato-mGFP mice with AAV8-TBG-Cre marks hepatocytes with GFP,intraperitoneal injection of carbon tetrachloride twice a week for two weeks or giving subtotal hepatectomy removeing about 70% of liver constructs acute liver injury models.Observe if there are any changes in the red and green fluorescence labeled areas and Immunofluorescence tests HNF4? expression and co-labeling with red or green fluorescence.3.Clarify whether hepatocytes can dedifferentiation into HPCs in chronic liver injuryContinuing to give the mice diet with 0.1% DDC for 4-12 weeks,immunofluorescence test PCK,EpCAM,Sox9 expression and co-labeling with red or green fluorescence.Repeat the above research in mice giving CDE diet.Separating the primary hepatocytes of mice wih DDC diet,collecting RNA and protein,RT-PCR and Western blot detect the expression of mature hepatocytes and HPCs related genes.4.Clarify the contribution of HNF4?in dedifferentiation of hepatocytesSeparating the primary hepatocytes of mice wih DDC diet weekly,collecting RNA and protein,RT-PCR and Western blot detect the expression of HNF4? each time.Cross mTomato-mGFP mice with HNF4? flox/flox mice,tail intravenous the new transgenic mice with AAV8-TBG-Cre knockouts HNF4? in hepatocytes specifically,immune fluorescence detects the expression of Sox9 in liver tissue.Separating the primary hepatocytes of mice,collecting RNA and protein,RT-PCR and Western blot detect the expression of mature hepatocytes and HPCs related genes.?Results?1.The generation of hepatocytes specially expressing green fluorescent protein(GFP) transgenic mice Tail intravenous mTomato-mGFP mice with AAV8-TBG-Cre,Immunofluorescence results showed that all hepatocytes expressed green fluorescence but other cells red fluorescence.Immunofluorescence detects the markers of other cells in liver,such as PCK,Sox-9,GFAP,alpha SMA,F4/80,CD34,the results revealed that in addition to hepatocytes,any other cells including biliary epithelial cells,hepatic stellate cells,Kupffer cells and vascular endothelial cells did not express green fluorescence.2.Self-replication is the only source of newly producted hepatocytes in acute liver injuryTail intravenous mTomato-mGFP mice with AAV8-TBG-Cre marks hepatocytes with GFP,giving intraperitoneal injection of carbon tetrachloride or 70% subtotal hepatectomy,immunofluorescence found that the areas of red fluorescence marked had no obvious expansion,and all HNF4? positive cells expressed green fluorescent but red fluorescence.Indicating self-duplication is the only source of newly produced hepatocytes in acute liver injury.3.Hepatocytes dedifderentiation to HPCs in chronic liver damage3.1 Continuing to give the mice diet with 0.1% DDC for 4-12 weeks,we found that,as the pastof time,the PCK(+)cells increased gradually and from DDC diet 6weeks on there started to appear green fluorescence marked PCK(+)cells.To detect other HPCs markers we also found green fluorescence marked Sox9(+)or EpCAM(+)cells.Illustrating that hepatocytes can dedifferentiation to HPCs in chronic liver injury of DDC diet.3.2 Giving the common C57 mice with DDC diet for 4 weeks.Separating the primary hepatocytes of mice wih DDC diet weekly,collecting RNA and protein,RT-PCR found that the expression of mature hepatocytes related genes reduced gradually and the expression of HPCs related markers increased significantly.Western blot revealed the expression of Sox9 also increased gradually with the time of DDC diet extending.These results confirmed the dedifferentiation of hepatocytes in RNA and protein level separately.3.3 Tail intravenous injecting mTomato-mGFP mice with AAV8-TBG-Cre marks hepatocytes with GFP,giving the mice with CDE diet,we also observed that with the time of CDE diet extending,the red fluorescence labeled areas expanded gradually.There also appeared green fluorescence marked Sox9(+)or PCK(+)or EpCAM(+)cells in CDE diet 6weeks.Indicating the dedifferentiation of hepatocytes still exist in CDE diet caused chronic liver injury.4.Knockouting HNF4? specifically leads to hepatocytes dedifferentiation spontaneously4.1 Giving the common C57 mice with DDC diet for 4 weeks.Separating the primary hepatocytes of mice wih DDC diet weekly,collecting RNA and protein,RT-PCR and Western blot test found that as the extension of DDC diet the expression of HNF4? reduced gradually,indicating that HNF4? may contribute to the dedifferentiation of hepatocytes.4.2 Cross mTomato-mGFP mice with HNF4? flox/flox mice,tail intravenous injecting the new transgenic mice with AAV8-TBG-Cre knockouts HNF4? in hepatocytes specifically,immunofluorescence test found that about 50% of the hepatocytes knocked out of HNF4? expressed Sox9 two weeks after the injection of AAV8-TBG-Cre.Separating the primary hepatocytes of mice,collecting RNA and protein,RT-PCR test found that the expression of most mature hepatocytes related genes reduced and the expression of most HPCs related markers increased significantly.Western blot revealed the expression of Sox9 also increased after HNF4? was knocked out specifically in hepatocytes.Indicating that HNF4? plays a great role in maintaining the function of mature hepatocytes and the downregulation of HNF4? expression is one of the important mechanisms of hepatocytes dedifferentiation.?Conclusions?1.Self-replication is the only source of newly producted hepatocytes in acute liver injury2.Hepatocytes can dedifderentiation to HPCs in pathologic conditions3.HNF4? plays a great role in the dedifferentiation of hepatocytes...