| Background:Partial hepatectomy (PH) is an efficient way to cure a variety of benign and malignant liver disease at present. However, when the PH range is too large or the liver itself is in a serious pathological condition, it’s easy to lead to refractory liver failure, which is a worldwide problem. Although liver transplantation can be an effective solution to the problem, but it makes liver transplantation is difficult to carry out because of the problem such as lack of donor or expensive. Besides, the existing bioartificial liver technology is not yet mature, and the efficacy of liver cell transplantation in vivo is limited. Therefore, lots of doctor want to solve the problem. Liver has a strong regeneration capacity, and liver cells are able to rapidly proliferating after PH, but its own regulatory mechanism is not clear right now. If we understand the mechanism, even if there is a huge extent of surgery or liver is in a serious pathological condition. So, it’s extremely important to explore liver regeneration self-regulation mechanisms.Our previous studies have shown that Tmubl (transmemebrane and ubiquitin—like domain containing 1) plays an important negative regulatory role in hepatocyte proliferation process. Tmubl was first reported by Fazia et al, and it played an important role in the process of proliferation of hepatocytes in 2005. Currently, The expression of Tmubl is high in the process of liver regeneration and in the nervous system, however, the role is not very clear. In the process of liver regeneration, Tmubl, upregulated and playing a negative role, can effectively prevent the excessive proliferation of liver cells. In the nervous system, Tmubl is highly expressed in the brain, conducive to recycle AMPAR (Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors) subunit GluR2(Glutamate Receptor 2) between the intracellular compartment and the cell surface, and it can interact on CAML (Calcium modulating cyclophilin ligand) involved in spontaneous activity and awakening. In short, the results show that Tmub1 gene and its prote play an important role in the process of liver regeneration, but the mechanism is not clear.Objective:1. To observe the impact of knocking down the expression of Tmubl on hepatocyte proliferation cycle and liver regeneration of normal rats after PH.2. To analysis possible relevant regulatory proteins, explore the regulatory mechanism of Tmubl on hepatocyte proliferation.3. To know whether the expression of Tmubl is abnormal or not in liver cirrhosis conditions, and evaluate its significance.4. To analysis the impact of abnormal changes of Tmubl on liver function and liver cell proliferation, and explore its mechanism.Methods:1. Influence of Tmubl gene silencing on the liver regeneration of normal rat after PH1.1 Rats experiment and grouping. Experimental conditions:male Sprague-Dawly rats, weighing 200g to 300g, placed under standard conditions:12h day alternately, to keep the room temperature constant, and free access to water and food, to ensure that there is no significant difference in weight and living habits of the rats in each group. Arrange 54 rats in three groups randomly, named Tmub1 RNAi experimental group, lentivirus empty vector group, and control group, and respectively select 3 rats at 6 time phase points like Oh,2h, 6h,12h,24h and 48h after PH in each group.1.2 Establish classical 70% rat PH model.1.3 Orthotopic liver cell separation and primary hepatocyte culture.1.4 Assay Tmubl mRNA and securin mRNA level by RT-PCR.1.5 Assay the expression of Tmubl protein and securin protein by Western Blot.1.6 Detect the liver cell proliferation by MTT.1.7 Observe the cell cycle by flow cytometry.2. Influence of Tmubl gene silencing on the liver regeneration of rat with liver cirrhosis after PH2.1 Rats experiment and grouping. Experimental conditions:male Sprague-Dawly rats, weighing 200g to 300g, placed under standard conditions:12h day alternately, to keep the room temperature constant, and free access to water and food, to ensure that there is no significant difference in weight and living habits of the rats in each group. Arrange 9 male SD rats with liver cirrhosis randomly in three groups, named Tmub1 RNAi experimental group, lentivirus empty vector group, and control group, besides another three normal male SD rats were used as the standard control group.2.2 Establish liver cirrhosis rat model.2.3 Establish classical 70% rat PH model.2.4 Test liver function in order to know the degree of cirrhosis and liver function status.2.5 Assay liver cell proliferation by Ki67 immunohistochemistry.2.6 Detect Tmubl protein level in different conditions by Western blot.2.7 Detect Tmub1 mRNA, IGF1 mRNA, HGF mRNA, TGF-betal mRNA, C/ EBP-beta mRNA, STAT3 mRNA, WT-1 mRNA level under different experimental conditions by Q-PCR.Results:1. Influence of Tmubl gene silencing on the liver regeneration of normal rat after PH1.1 Tmubl RNAi interference effect detectionThe expression of Tmubl mRNA and protein were significantly lower in experimental group.1.2 The impact of Tmub1 knocked down on liver cell proliferationMTT shows that liver cell proliferation rate can be significantly up-regulated by knocking down the expression of Tmubl after PH 6h-24h.1.3 The impact of Tmub1 knocked down on securin mRNA and its proteinThe expression of Securin mRNA has no significant change, but in the G2/M phase securin protein was significantly reduced.2. Influence of Tmub1 gene silencing on the liver regeneration of rat with liver cirrhosis after PH2.1 Observing the cirrhotic rats pathologyUnder a microscope, the normal lobular architecture was destruct, fibrous connective tissue dysplasia, and a wide range of false lobules were formed.2.2 Tmub1 RNAi interference effect detectionThe expression of Tmub1 mRNA and protein were significantly lower in experimental group.2.3 Detecting the difference between normal rats and cirrhotic rats of the expression of Tmub1 protein by western blot and Q-PCRThe Tmub1 gene and protein of cirrhotic rats were significantly increased comparing with normal rats.2.4 Differences in detection of liver function in different experimental modelsLiver function of Tmub1 RNAi group was significantly improved comparing with the cirrhosis group and lentivirus empty vector group.2.5 Assay of the liver cell proliferation by Ki67 immunohistochemistryKi-67 positive rate Tmub1 RNAi group was significantly raised compared with the cirrhosis group and lentivirus empty vector group. That means that knocking down the expression of Tmub1 can effectively promote liver regeneration rate after PH in liver cirrhosis condition.2.6 Detecting IGF1 mRNA, HGF mRNA, TGF-beta1 mRNA, C/EBP-beta mRNA, STAT3 mRNA, WT-1 mRNA level under different experimental conditions by Q-PCRIGF1 mRNA, HGF mRNA, TGF-beta1 mRNA, C/EBP-beta mRNA, WT-1 mRNA was significantly increased, STAT3 mRNA was significantly reduced comparing with normal rats in our liver cirrhosis model. And after knocking down the expression of Tmub1, each factors almost returned to normal.Conclusion:1. Lentivirus vector is suitable for the target gene RNA interference experiments, even it can also play a better effect in vivo.2. Tmub1 protein may play an important role in the liver cell cycle G2/M phase.3. Tmub1 protein playing an important role in the process of liver regeneration may be closely related to affect the expression of Securin at the protein level.4. Tmub1 gene and protein in rats with liver cirrhosis were significantly increased comparing with normal rats.5. Knocking down the expression of Tmub1 can improve the liver function and liver cell proliferation of cirrhotic liver rats after PH.6. In our liver cirrhosis model, IGF1 mRNA, HGF mRNA, TGF-beta1 mRNA, C/EBP-beta mRNA, WT-1 mRNA was significantly increased, STAT3 mRNA was significantly reduced comparing with normal rats. And after knocking down the expression of Tmub1, each factors almost returned to normal. This may be an important reason why reducing Tmubl expression can improve liver function and the ability of liver regeneration in liver cirrhosis condition after PH. |
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