Research Of CCL2 And Its Receptor CCR2, And S100A11, S100A6 In Regulating Liver Regeneration

As the biggest digestive organ in body and the most important site of metabolism, liver is the only organ which has the ability of regeneration. Under normal condition, most hepatic cells are in quiescent stage of cell cycle, and only 0.001~0.01 percent of hepatic cells can replicate DNA regularly. When liver is damaged or surgically and partially removed, most hepatic cells will enter into G1 stage from quiescent stage, and proceed to mitosis. Then cells proliferate largely and liver recovers as before. This is the whole process of liver regeneration. Research on the mechanism of liver regeneration is helpful for patients who have liver disease. Presently, the pathway of liver regeneration can be divided into three networks, including cytokine network, growth factor network and metabolism network, which interact with each other and regulate liver regeneration in concert. But the mechanism of liver regeneration is still a puzzle, whose regulatory process can not be fully expatiated. The aim of this research is to identify new functional genes in regulating liver regeneration, and to provide new clue and theory for exploring novel liver-protecting drugs.The foundation of this research is the result of gene chip study conducted in our lab, which reveals the changes of genome transcripts after mouse liver damaged by CCl4. We selected four genes which are up-regulated after the acute liver damage, including chemokine CCL2 and its receptor CCR2, and Ca2+-binding protein S100A11, S100A6. We plan to construct the pcDNA3.1 eukaryotic expression vector of CCL2, S100A11 and S100A6, and to use these vectors for in vivo animal studies through electroporation to initially verify the function of CCL2, S100A11 and S100A6 in regulating liver regeneration. We also plan to construct the prokaryotic expression vector of the first 55 amino acid of CCR2, which could bind to CCL2, and express and purify the recombinant protein to prepare rabbit polyclonal antibody. We will do antibody block experiment for identifying the role of CCR2 in liver regeneration. Then we will construct the prokaryotic expression vector of CCL2, S100A11 and S100A6, induce, express and purify the recombinant proteins which have biological activities for further identifying the function of the proteins in regulating liver regeneration.Using electroporation, we found CCL2 could cause further damage of liver induced by CCl4, which displayed larger and fatty hepatic cells, shrinked cholecyst and intumescent stomach. We think it is hepatitis. After we found CCL2 could inhibit the liver regeneration, we decided to use CCR2 antibody to block the binding between CCL2 and CCR2, to identify the function of CCR2 in liver regeneration. We used pGEX prokaryotic expression vector, expressed and purified the first 55 amino acid of CCR2. Then we prepared rabbit polyclonal antibody, and determined the potency of antibody by Western blot and ELISA. According to the result of antibody block experiment, we found the activity of aminotransferase between experiment group and control group had no difference, which suggests that the first extracellular region of CCR2 may not be involved in liver regeneration.We constructed the pET-28 expression vector, after induction the protein was found in inclusion bodies. We used 8M urea to denature the protein then purified it by affinity chromatography followed by renaturization. Finally we obtained very pure CCL2 recombinant protein, which provided the base for further research of CCL2 in liver regeneration.After transferring pcDNA3.1-S100A11 plasmid DNA into mouse via in vivo electroporation, we found at the fourth day after liver damage, the activity of aminotransferase in the serum of experiment group mouse is higher than that of the control group mouse. The HE staining result of liver damage with mouse liver section displayed the hepatic cells became vacuolar, ruleless and fat deposition. The PCNA immunohistochemical staining result of liver damage with mouse liver section indicated that the experiment group still had proliferating cell, whereas the control group had completed liver regeneration. So we elicited S100A11 could inhibit liver regeneration and slower the proliferation of hepatic cells. Then we constructed the pET-28 prokaryotic expression vector of S100A11. After induction and expression, we found the protein was in soluable form. With affinity chromatography, we recovered the pure S100A11 recombinant protein.After pcDNA3.1-S100A6 plasmid electroporation, we found the experiment group mouse had stronger tolerance to toxicity induced by CCl4 than the control group mouse. At the fourth day and seventh day after liver damage, the activity of aminotransferase in the serum of experiment group mouse is lower than the control group mouse. The HE staining result of liver damage with mouse liver section displayed the liver recover of experiment group mouse is better than the control group mouse. And the result of PCNA immunohistochemical staining indicated that the experiment group had completed liver regeneration, while the control group still had proliferating cells. Therefore, we concluded that S100A6 could be helpful for hepatic recover after liver damage.All these studies highly suggested that chemokine CCL2, calcium-binding protein S100A11 and S100A6 played roles in regulating liver regeneration. And we expressed and purified CCL2 and S100A11 recombinant protein, constructed prokaryotic expression vector of S100A6, which will supply groundwork for farther research on the effect and mechanism of those proteins in liver regeneration. These results can also provide useful information for systematic research on signal transduction networks controlling liver recovery and regeneration processes. Furthermore, the inter-relationship between chemokine family and calcium-binding protein family may open a new window for studies in the field of liver regeneration.