| Liver is an important organ for nutrient metabolism. Liver regeneration, which is initiated by hepatocyte injury in variou diseases, is mediated by several types of stem cells. Liver stem cells include endogenous stem cells and exogenous ones. The endogenous stem cells are mainly hepatic oval cells, while the major exogenous stem cells are hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor stem cells, etc. Hepatic oval cells play a crucial role in liver regeneration. They are pluripotent cells which distributed along with the Hering canal of intrahepatic bile duct. Although the oval cells are static under normal circumstances, they are activated by stimuli to proliferate and differentiate into bile duct cells and hepatocytes, thereby contributing to morphological and functional repair of the organ. Up to now, the mechanisms regulating proliferation and differentiation of oval cell is elusive, we therefore investigated the potential regulation mechanism.According to the recent research, DNA methylation of epigenetic is closely related with the proliferation and differentiation of stem cells. The DNA hydroxylase TET1(Ten-eleven translocation1) is the key factor that convert5-methylcytosine into5-hydroxymethylcytosine, which plays a dual role of promoting the transcription of sternness gene and inhibiting its differentiation of gene expression during the embryonic stem cells differentiation process. Meanwhile, TET1is down-regulated in the development of embryonic stem cells. However, the specific mechanism of TET1regulating self-renewal and differentiation of stem cells is not clear.Consequently, we have designed our resesch as follows:1. Establishing the animal model of oval cell proliferation successfully.2. Isolation, extraction, culture and identification of primary oval cells of rat. To obtain primary oval cells of rat and identify its specificity.3. Detection of the dynamic expressions of TET1in the animal model of liver regeneration. Exploring the expression changes of TET1during liver regeneration at different times through different experimental methods.4. Detecting the expression changes of TET1during the hepatocytic differentiation process of the oval cell line WB-F344. The gene and protein levels of TET1can be analyzed by establishing the model of oval cell differentiated into hepatocyte-like cells in WB-F344oval cell line. To sum up, we illustrated that:1. TET1showed a significant changes during the dynamic process of liver regeneration.2. The expression level of TET1was significantly decreased in the differentiation of oval cells into hepatocyte-like cells, which preliminarily suggested that TET1may be involved in the process of proliferation and regeneration of oval cells.Method1. Establishment of the animal model of liver regeneration. SD rats, weighted approximate150-180g, were administrated with2-AAF10mg/kg·d for4days and underwent surgical resection of70%liver on the fifth day, then administrated with2-AAF continuously. Acquiring specimens of liver on the3rd,6th,9th,12th and15th after liver resection.2. Isolation and identification of primary oval cells of rat. Primary rat oval cells were isolated by collagenase perfusion, then purified by Percoll densitygradient centrifugation. Identification methods including the morphology, cell specific markers of hepatic oval cell, ect.3. Detecting the dynamic expressions of TET1in the animal model of liver regeneration. TET1was detected in the mRNA levels of RT-PCR and in the protein level of Western blot at different times.4. Numbers of cytokines induced oval cell differentiated into hepatocyte-like cells. Several cytokines were applied to induce hepatocytic differentiation of oval cells. The experiment was divided into two groups. The cells in the control group was cultured without cytokines for24h, while the cells in the experimental group was cultured with SCF20ug/L, EGF10ug/L, and HGF10ug/L for7days.5. Detecting the specific marker of oval cell and specific protein of hepatocyte in WB-F344cells before and after induction. Detection of oval cells before and after induction by immunofluorescence, including the specific OV6antibody of oval cell, the specific ALB and AFP antibody of hepatocyte and the specific CK19antibody of bile duct epithelial cell.6. Analysis of TET1expressions in oval cells before and after induction by immunofluorescence, Realtime PCR and Western blot.. Results1. The animal model of oval cell proliferation was established successfully. The remnant liver was collected on the3rd,6th,9th,12th and15th day after resection. It could be found that the abdominal incision of rat was gradually healed while the volume of remnant liver was gradually increased until the liver restoring to the normal size on the15th day.2. We obtained oval cells up to1.34×105/ml. Objective cells seen under the inverted microscope extraction are spherical, which were patially adhered to the dish bottom after culture for24h. Immunofluorescent staining showed that the oval cell expressed specific markers OV6,with a purity≥80%.3. TET1expression was analyzed by RT-PCR and Western blot in the liver tissues of the animal model. Compared to control, TET1expression was significantly increased in the3d and6d group (P<0.01). No statistical difference was identified between the9d group and controls (P>0.05). However, TET1expression was downregulated at the later stage (12,15d) of liver regeneration (P<0.05).4. Compared to control group, the morphology of oval cell line WB-F344has developed into long fusiform or polygonal cells and the nucleo cytoplasmic ratio was decreased after induction by SCF20ug/L, HGF lOug/L and EGF10ug/L, which were similar to hepatocytes in vitro.5. After induction by SCF, HGF, and EGF, WB-F344cell stained negative for OV6and positive for ALB and AFP, while the control cells were OV6positive, and ALB and AFP negative..6. The mRNA level of TET1was down-regulated in oval cells after induction by SCF20ug/L, HGF10ug/L, EGF10ug/L (P<0.01). Immunofluorescence and Werstern blot also showed a decrease protein level of TET1, which was in according with the gene expression level.Conclusion1. The animal model of oval cell proliferation has been established successfully by2-AAF combined with2/3liver resection. 2. Plenty of hepatic oval cells with high purity could be obtained by primary cell extraction in the model of liver regeneration, which suggested that a large number of oval cells proliferated in the process.3. Expression of TET1significantly increased in the early stage of the liver regeneration animal model, until the peak on the sixth day after liver resection, then the expression of TET1gradually decreased while the liver was recovering. The result indicated that it was similar to the proliferation of oval cells in the liver regeneration process, which was proved that TET1was closely related with the proliferation process of oval cells.4. During the process of oval cells proliferating into hepatocyte-like cells in vitro, expression of TET1showed a down-regulation while the stemness of oval cells decreased, which indicated that TET1was closely related with the self-renewal and differentiation of oval cells, thus TET1may be involved in the liver regeneration process by regulating the differentiation of oval cells. |
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