| Background :In recent years, due to changes in lifestyle and the deterioration of living environment, various liver diseases in our country is on the rise. At present, the most effective treatment of a variety of benign and malignant liver disease is still The PH(Partial Hepatectomy). Although liver transplantation is an effective treatment, the shortage of donor limits the extensive liver transplant. The existing biological artificial liver technology still cannot completely replace the liver function. Hepatocyte transplantation in vivo after proliferation is limited, also limits its therapeutic effect. But, unlike other organs, liver regeneration ability has great potential, therefore, to explore the liver cell regeneration molecular mechanism has very important practical significance, thus for clinical promote liver regeneration, and the theoretical basis for the prevention and treatment of liver failure.Generally accepted at present is that the residual liver regeneration after PH is not through the liver stem cells, but through the remaining hepatocytes proliferation. Hepatocyte proliferation cycle involving many cytokines and growth factors involved, such as IL- 6, HGF and TGF- alpha, etc. Literature reported that about 60% of the PH after 24 hours of liver cells are completed a cell proliferation cycle[1-5], but only a small number of liver cells can enter the second cell proliferation cycle. Why liver cells will automatically terminate after completing one proliferation cycle, the current research on its regulatory mechanism is few, especially for liver cell proliferation how to enter the cell cycle G2 / M phase, and by what factors control, the current lack of in-depth study.Tmub1 protein shuttles between the nucleus-cell plasma, it contains the ubiquitin like domain, the area contains UCH, E2 and CUE reaction sites. Tmub1 protein almost all exists in the nucleus in the late liver cell proliferation cycle. In the nucleus, before sister chromatids separation by adhesive proteins, cohesin complexes are linked together, under the control of the activity of separase, and the activity of separase controlled by securin, a protein of 202 amino acid residues, mainly in the cytoplasm, only a fraction is located in the nucleus[6]. In the middle of the M phase, securin degradated by ubiquitin- proteasome pathway under the participation of APC, separase this release, activation, cohesin complexes are broken down, sister chromatids separation, so as to complete a proliferation of cell division[7].Tmub1 shuttles into the nucleus in G2 phase, and this is the proliferation of cell mitosis run-up, and when the cells into G1 phase, Tmub1 expresses in cytoplasm is almost completely[3-5]. Securin protein and Tmub1 protein, therefore, could be combined because of their complementary structure and their space is similar. If we could prove that Tmub1 combines with securin and affect liver cell proliferation, would provide a new basis in the process of liver regeneration and liver cell proliferation regulation, it has very important clinical significance.Objective:1. To identify Tmub1 protein combined with securin protein after hepatectomy.2. To know the role of Tmub1 protein in liver cell proliferation.Methods:1.(1) A total of 40 adult male S-D rats were randomly divided into 5 groups, each group of 8, the first group of rats had been done PH, then directly took the original generation liver cells after liver perfusion. The rest of the four groups had been done PH, in postoperative 12, 24, 48, 72 h took the original generation hepatocytes respectively.(2) Each group of liver cells after cracking to extract protein.(3) Part of protein did immune coprecipitation experiments first, and then did western-blot experiments to know Tmub1 protein combined with securin protein and its change trend with time after partial hepatectomy; Another part of directly for western-blot experiments, to understand the change trend of intracellular Tmub1 after liver resection.2.(1) Build expression Tmub1 Lentivirus vector and a negative carrier control.(2) Build silent Tmub1 Lentivirus vector and negative carrier control.3.(1) Using silence Tmub1 virus vector, expression Tmub1 virus vector and empty vector to transfect rats BRL – 3A liver cell lines.(2) Determined by MTT and flow cytometry test the cell cycle after transfection, compared between groups, understand the role of Tmub1 protein in liver cell proliferation.Results:1. Established a stable model of 70% hepatectomy in SD rats.2. Through the Co-IP technology confirmed Tmub1 protein combined with securin protein after hepatectomy. The western blot after Co-IP get securin protein bands show Tmub1 proteins and securing proteins after liver resection combined with each other. The result of the strip, The grey value of 0h after hepatectomy was low, as the time increases gradually increased after hepatectomy, the 48 h was highest, 72 h fell again.3. The Tmub1 protein western blot after liver resection detected that each phase Tmub1 protein content was not obvious change. Directly Tmub1 protein western blot showed that each phase after hepatectomy with the same brightness, IMAGE J image software analysis each gray value of all groups, and statistical analysis, there was no significant difference of the Tmub1 protein content of every phase after hepatectomy(p > 0.05).4. Successfully constructed the lentivirus vectors with Tmub1 gene overexpressed, and obtain the corresponding virus, the virus titer was 2 × 108 TU/ml. Successfully constructed the silent Tmub1 lentivirus vectors, obtain the corresponding virus, the virus titer was 7 × 108 TU/ml.5. Through LV-Tmub1, the negative control vector of LV-Tmub1 and LV-Tmub1-RNAi, the negative control of LV-Tmub1-RNAi transfection BRL-3A cell lines, western blot detection Tmub1 protein bands found LV-Tmub1 expression was obviously enhanced, LV-Tmub1-RNAi group was obviously reduced, the IMAGE J image analysis software analysis gray value of every group,and statistical analysis, Tmub1 protein content of the LV-Tmub1 and LV-Tmub1-RNAi groups was statistically significant(p < 0.01).6. MTT experiments to detect cell proliferationLV-Tmub1,the negative control vector of LV-Tmub1 and LV-Tmub1-RNAi,the negative control of LV-Tmub1-RNAi transfected BRL-3A cell lines,and normal BRL-3A cell lines,MTT experiment compared all groups after 48 hours cultivation,found that the OD value of the group of LV-Tmub1 transfection of BRL-3A cells is significantly higher than the group of LV-Tmub1-RNAi transfected BRL-3A cell lines.7. Flow cytometry to detect the cell cycleThough LV-Tmub1,the negative control vector of LV-Tmub1 and LV-Tmub1-RNAi,the negative control of LV-Tmub1-RNAi transfected BRL-3A cell lines,and normal BRL-3A cell lines,flow cytometry instrument detected cell cycles,found the group of LV-Tmub1 transfection of BRL-3A cells in the G2+S phases is significantly higher than normal cell lines,and the group of LV-Tmub1-RNAi transfected BRL-3A cell lines in the G2+S phases is lower than normal cell lines.Conclusion:1. The Tmub1 protein combined with the securin protein after hepatectomy.2. Tmub1 begin to flow from the cytoplasm into the nucleus 12 hours after hepatectomy, and combined with securin protein, after 48 hours to reach peak and then gradually decline. Tmub1 protein itself content in the hepatocytes has not obvious change.3. Tmub1 protein could promote the hepatocyte proliferation. |
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