A Study Of Liver Damage Repair And Liver Tumorigenesis In HSV-tk Transgenic Mouse Model

The liver has pivotal role in body homeostasis,a wide variety of insults can affect the liver leading to development of steatohepatitis,fibrosis,cirrhosis,cancer and finally death.Hepatocellular carcinoma(HCC)is the second most common cause of cancer-related death in the world,whose pathogenesis is incompletely understood.To clarify the pathogenesis of HCC and investigate the effects of potential therapies,a number of mouse models have been developed.Genetically engineered mouse models have greatly facilitated studies of gene function in HCC development.There is no doubt that these genetically engineered mice are valuable tools for understanding the underlying biological mechanisms of tumorigenesis.Liver diseases have inflammation and fibrosis as precursors to cancer,yet none is easily mimicked in animals.It is important to obtain mouse model that can imitate the complex process of human liver damage eventually lead to liver cancer.We previously generated a transgenic HSV-tk mouse using pronuclear microinjection,in which HSV-tk was driven by a serum albumin promoter?enhancer.We further get three lines of transgenic HSV-tk mouse and investigated intensively the integration sites of HSV-tk by using fluorescence in situ hybridization(FISH)and inverse PCR(i PCR).The results demonstrated that HSV-tk was integrated into chromosomal sites8C3-C4,5E1-E2 and 11A1-A2 in three transgenic mouse lines,respectively.Among these integration sites,8C3-C4 was located in intergenic region,while the other two were in introns of Rufy3 and Abca13,respectively.Immunofluorescence staining showed different levels of HSV-tk expression in livers of three transgenic lines.After administration of GCV,chronic hepatitis pathological changes in livers were observed.Immunohistochemical staining showed significantly increased of the cells with proliferating marker Ki67~+,cholangiocytes marker CK19~+and?-SMA~+,a marker for activated hepatic stellate cells in liver related to ductular reactions observed in chronic liver diseases.In addition,blood test demonstrated a significantly increased serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST).We used transgenic HSV-tk mouse as a GCV-induced liver injury model to study the repair effect of in utero injection of hematopoietic stem cells(HSC)on hepatic damage.The results showed that under the conditions of hepatic damage HSC can be homing to the liver and proliferation and differentiation,thus results to a certain extent of liver damage protection and repair effect.Interestingly,three lines of transgenic HSV-tk mouse have developed of liver cancer after age of six months even without GCV administration.To address this question,we analyzed the transcriptome of HSV-tk hepatocarcinoma and hepatitis using microarray analysis to investigate the possible causes of hepatocarcinoma.GO enrichment analysis showed that the up-regulated genes of hepatocarcinoma mainly include the immune and cell cycle genes.The dysfunction of a large number of immune-related genes indicates that the immune system plays an important role in the occurrence and development of HSV-tk related hepatocarcinoma.The down-regulated genes in hepatocarcinoma are mainly concentrated in the lipid metabolism related genes,which make the cells to have fatty degeneration.HSV-tk murine hepatocarcinoma was compared to published Notch murine hepatocarcinoma.Gene set enrichment analysis(GSEA)was used to compare the up-regulated and down-regulated genes and signaling pathways,showing that in the HSV-tk mouse immune-related signals are up-regulated compared to Notch mice,while genes involved in cell cycle and DNA repair pathways were down-regulated.This suggests that increased expression of immune-related genes is characteristic of HSV-tk mice.The above analysis suggests the complex causal relationship between the inflammatory—cell cycle activated—hepatocarcinoma cycles.By analysis the TCGA database of human liver cancer,we found that the signaling pathways of immune/inflammatory reaction in human liver with high heterogeneity,only high expression in some samples.In this study,we use HSV-tk transgenic mouse as a GCV inducible liver injury mouse model in the study of in vivo damage repair function of hematopoietic stem cells.Transcriptome analysis of HSV-tk tumors revealed that immune system and lipid metabolism disorders might plays an important role in the hepatocarcinoma occurrence.It develops a reliable mice model and provided experimental data basis for hepatocarcinoma and hepatic damage which can be significantly helpful in clinical research and potential application in liver specific diseases.