| The standard treatment for end-stage liver disease is liver organ transplantation.However,due to the shortage of donor liver,or the death of patients before transplanted the suitable liver,hepatocyte transplantation has been emerging as an alternative treatment for patients with liver disease,liver cells are transplanted into the human body to replace the damaged liver.It has been developed for more than 30 years,there are still some problems such as difficulty in proliferation of transplanted cells in vivo.In our study,we noticed that the process of liver cell regeneration was the result of the interaction between hepatocytes and liver microenvironment.The major issue of hepatocyte transplantation in clinical application is absence of hepatocytes with good quality,most of researchers have been looking for alternative cell sources,they found that some stem cells could differentiate into liver cells with function,including somatic cell induced pluripotent stem cells?i PSC?,embryonic stem cells?ESC?,mesenchymal stem cells?MSC?and so on.Although these hepatocyte-like cells could achieve large-scale proliferatation in vitro and express specific genes of mature hepatocyte after differentiation,the function of those cells are unstable,and the efficiency of reconstruction in vivo is very low.Further more,there was a high security risk in the clinical application of virus infection and gene recombination.Thus we have been trying to use small molecules to induce primary hepatocyte transdifferentiation.After a long time of exploring and screening,finally we defined a combination of small molecular compounds to achieve the proliferation of mouse primary liver cell in vitro.The proliferating hepatocytes was named as hep PDCS,with the characteristics of liver progenitor cells and the ability to differentiate into mature hepatocytes?named as hep PDCS-hep?.Compared to other liver cell resources,it has no any infection by the virus,the genome has not inserted any fragments,which ensure the safety of clinical application.In addition,hep PDCS-hep could replace the injured mouse liver with high replacement rate with an efficiency of 70%,suggesting good medical application prospects.Meanwhile,we investigated the effect of microenvironment of liver injury on liver transplantation.Liver microenvironment mainly consists of non parenchymal cells,such as macrophages,sinusoidal endothelial cells,stellate cells,and various cytokines and matrix secreted by cell.Among them,the number of macrophages accounts for 15% of the total number of liver cell.There were numerous studies indicated that macrophages in liver had a wide range of biological functions: antigen presentation,phagocytosis,participating in liver metabolism,contributing to the development of tumor and promoting liver regeneration and so on.Activated macrophages secrete many cytokines such as TNF,IL-1,IL-6,TGF-B,of which TNF-A and IL-6 can start up the process of liver regeneration and activate the proliferation of hepatic stem cells.Subsequent studies suggested that the IL-17 secreted by macrophages also played an important role in the process of liver regeneration.Some researchers have shown that the removal of macrophages in the model of liver resection would delay the process of liver regeneration,while it also said that macrophages deletion promoted liver regeneration.However,no study has been conducted to investigate whether macrophages have an significant effect on hepatocyte transplantation.We hereby ensumed that macrophages might play an important role in hepatocyte transplantation in Fah deficient mouse modle.The role of macrophages in hepatocyte transplantation can help us understand the process of liver regeneration and find way to improve the efficiency of hepatocyte transplantation.In summary,through the reprograming of mature hepatocytes,we obtained a novel cell source with good function and proliferation ability,via exploring the changes of liver microenvironment in the process of liver generation,we found effective way to improve the efficiency of liver cell transplantation,so as to provide reference for new strategies and clinical study on hepatocyte transplantation.?Methods?1.The screening of small molecular compounds for hepatocytes expansion is determined by gene reporter system based on CK19-Cre ERT/R26 GFP mouse.2.To identify the reversible transition between hepatocytes and duct-like progenitor cells,we use the testing methods of immunofluorescence,flow cytometry,microarray analysis,PCR;Then we assess whether liver cells have the capacity to large-scale proliferation with stable phenotype in vitro by the methods of cell counting,karyotype analysis;3.To exclude the possibility that the duct-like cells originated from other liver nonparenchymal cells during cell isolation,R26 YFP mice were infected with 1×1011AAV8-TBG-Creviral particles by intravenous injection,primary liver cells with GFP were sorted by flow cytometry after 1 week and then cultured in TEM.4.The bipotential differentiation of hep PDCs is measured by immunofluorescence,microarray analysis,Rho123 staining,drug metabolism,El ISA and so on.5.To test whether hep PDCs-derived hepatocytes could act as functional hepatocytes in vivo,the cells were transplanted into Fah-/-mice.After 2 month,cell engraftment area was measured by immunohistochemistry and biochemical test.6.The changes of macrophages were observed at different time points after the removal of the drug NTBC used to maintain the survival of Fah-/-mice.7.After deleting the macrophages in the Fah-/-mice with liposome,primary hepatocytes were transplanted into the spleen,proportion of the hepatocytes regeneration areas was measured and compared at different time points.8.After separating bone marrow mononuclear cells of Fah-/-mice,GM-CSF cytokine was used to stimulate monocytes into macrophages.By culture and identification,the purified macrophages and hepatocytes were mixed at a certain ratio and then transplanted into Fah-/-mice.?Results?1.Long term expansion of hepatocytes in vitro under the certain culture condition.2.Mature hepatocytes isolated from CK19-Cre ERT/R26 GFP mouse and R26 YFP mouse injected with AAV8-TBG-Cre can transfer to duck-like progenitor cells in vitro.3.Hep PDCS has the characteristic of liver progenitor cell: hep PDCS aquire hepatic differentiation ability after hepatic stimulation to express the genes related to hepatic function,secrete albumin,metabolize drugs,synthesis urea,storage glycogen,and differentiate into functional BECs in three-dimensional culture.4.Hep PDCS-hep can repopulate chronically injured liver tissue of Fah-/-mice and increase the survival rate of the mice,with an enfficiency to 90%.5.After the withdrawing of NTBC,the kupffer cells in liver of Fah-/-mice would be activated.6.Deletion of kupffer cells in liver advances the repopulation efficiency of Fah-/-mice.7.The co-injection of macrophages and hepatocytes aggravates the liver injury and reduces the repopulation efficiency of Fah-/-mice. |
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