| Objective:We want to investigate that the role of a new histone demethylase plays in liver cirrhosis. This helps us to understand the role of epigenetic molecules in human disease and explore the possibility of such molecule as a potential target in disease treatment.Methods:1. We tested the level of RBP2expressed in20pairs specimens (cirrhosis of the liver tissues and liver normal tissues) by Immunohistochemistrical detection and Identified the differences of R.BP2expressed in these tissues, then analyzed these results, which provided the foundation of the role of RBP2in cirrhosis.2. In the molecular level, we treated hepatic stellate cells by specific RBP2siRNA and then detected the expression of liver fibrosis associated α-SMA and vimentin. After that, we transfected the RBP2overexpression plasmid into cells, and determined the expression of α-SMA and vimentin, which helped us to identify whether RBP2regulated the above mentioned molecules. We also analyzed cell proliferation by colony formation assay after RBP2knocked out. In addition, we used some liver fibrosis induced factors such as TGF-β to treat cells and detected the expression of RBP2, α-SMA and vimentin. We first used specific RBP2siRNA to treat hepatic stellate cells and then used TGF-β to induce HSCs, and analyzed the expression of α-SMA and vimentin as well as the cell proliferation of HSCs, which helped us to identify that whether TGF-β regulated α-SMA and vimentin via RBP2. The followings are the specific methods in this part:(1) After RBP2siRNA and the control siRNA transfection into LX-2cells for72hours, we determined the expression of RBP2, a-SMA and vimentin.(2) After RBP2siRNA and the control siRNA transfection into LX-2cells for72hours, we determined the ability of these cells to form colones two weeks later.(3) After RBP2overexpression plasmid and its control plasmid transfection into LX-2for48hours, we determined the expression of a-SMA and vimentin.(4) We used TGF-βto treat LX-2,48hours later, we identified the expression of RBP2, a-SMA and vimentin.(5) We first used RBP2siRNA to treat LX-2and24hours later added TGF-β,we identified the expression of RBP2, α-SMA and vimentin48h later. Similarly, we detected the cell proliferation of LX-2by colony formation assay with the same ways.3. In the organic level, we divided15rats into3groups,9for the experimental group and6for the normal control group. The experimental rats drank water containing10%alcohol, and fed with high-fat and low-protein foods, meanwhile the rats were subcutaneous injected with the mixed solution (40%CCl4and60%of a mixture of olive oil) once every four days, and the dose was0.5ml/g. The normal control group drank water and subcutaneous injection with the same dose of saline. We weighed the rats every four days.6weeks later, we killed all these rats, dissected livers and spleens and weighed them. The livers were divided into two parts, one part was put in neutral formalin for HE and immunohistochemical detection and the other was frozen at-80degrees refrigerator for extracting RNA purposes.Result:1. The expression of RBP2in cirrhosis liver tissues was stronger than liver normal tissues, and immunohistochemical staining showed the number of RBP2positive cells increased significantly in the cirrhotic liver tissues.2. After RBP2siRNA transfection into LX-2cells for72hours, we determined the expression of RBP2, α-SMA and vimentin decreased significantly, which indicated that RBP2may participate in liver fibrosis and liver cirrhosis by regulatinga-SMA and vimentin.3. After RBP2siRNA and the control siRNA transfection into LX-2cells for72hours, we determined the ability to form colones in RBP2siRNA cells decreased compared with control group.4. After RBP2overexpression plasmid and its control plasmid transfection into LX-2for48hours, we determined the expression of RBP2, α-SMA and vimentin elevated.5. After using TGF-βto treat LX-2for48hours,we identified the expression of RBP2, a-SMA and vimentin increased significantly.6. We first used RBP2siRNA to treat LX-2and24hours later added TGF-p,we identified the expression of RBP2, a-SMA and vimentin as well as the proliferation of LX-2were attenuated.7. In the organic level, the liver tissues of the experimental group of rats appeared obvious cirrhosis characteristics. The experimental rats liver index (liver weight/body weight) and spleen index (spleen weight/body weight) were significantly higher than control group. RBP2mRNA and protein expression in the cirrhotic liver tissue of the experimental rats were significantly improved.Conclusion:Some cirrhosis induced factors, such as TGF-β may regulate the expression of cirrhosis associated molecules a-SMA and vimentin via RBP2to promote the occurrence of cirrhosis. RBP2may become a potential target for the diagnosis and treatment of cirrhosis of the liver. |
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