Tmub1蛋白在肝细胞增殖周期中对securin类泛素化的调控机制及其对肝再生的影响

Tmub1 protein plays an important role in regulating hepatocyte proliferation.We found that the rate of liver regeneration significantly increased in M phase by blocking Tmub1 after partially hepatectomized(70%).However,securin protein is an important factor in the process of segregation of sister chromatids in G2/M phase, and it is degradated by ubiquitin - proteasome pathway which is regulated by the ubiquitin molecule in the nucleus. In addition,tmub1 protein with UBL domain is abundantly expressed in the nucleus in the liver cell proliferation G2/pre-M phase. Therefore, we speculate Tmub1 protein may be involved in the ubiquitination-like regulation of securin protein,thus it can regulate chromosome segregation in mitotic and play an important regulatory role in liver cell proliferation cycle. Through 70% hepatectomy rat model, The relationship between the two proteins was analysised by CO-IP and FRET; whether the mode of action of the two protein were related to the ubiquitination degradation was detected by Mass Spectrometry and Ubiquitin fluorescent probes; chromosome segregation and proliferation process of hepatocytes were studied by FCM and microscopic chromosome separation technology. This subject is to study the molecular regulation mechanism in the hepatocyte proliferation after PH.It will provide a new mentality for studying the feedback control mechanism in liver regeneration and a new method forpromoting the remnant liver regenerationand the clinical treatment of liver failure.

Tmub1蛋白在肝细胞增殖中发挥了重要的调控作用。我们通过沉默Tmub1发现70%肝切除术后残肝细胞在G2/M期的增殖速率明显增加。而securin正是调控细胞M期染色体分离的重要因子，该蛋白在胞核内经泛素-蛋白酶体通路降解，并受到类泛素化分子调控；具有UBL结构域的Tmub1蛋白在肝细胞增殖G2期、M前期逐渐大量表达于胞核中，因此我们推测Tmub1蛋白可能参与securin类泛素化调控作用，影响分裂期染色体分离，从而对肝细胞增殖进程发挥重要作用。通过复制大鼠70%肝切除术动物模型，运用CO-IP、FRET等分析两蛋白间关系，质谱分析及泛素荧光探针检测两蛋白作用方式是否与泛素化降解相关，运用FCM及显微染色体分离技术研究肝细胞染色体分离及增殖周期进程。本课题旨在研究肝切除术后肝细胞增殖及分子调控机制，为探讨其自身调控机制提供一条新思路，从而为临床促进残肝再生、防治肝功能衰竭提供新方法。

Tmub1在肝细胞增殖中发挥了重要的调控作用，而其中的机制，尤其是Tmub1与细胞周期相关蛋白的泛素化，仍少有报道。目的：研究Tmub1在肝再生中的调控作用以及与securin的相互作用。方法：重复大鼠70%肝切除模型，观察肝再生全程Tmub1和securin的表达趋势，通过co-IP和免疫荧光研究二者的相互作用；使用大鼠正常肝细胞系BRL-3A构建Tmub1过表达和干扰慢病毒稳转株，使用Edu、CCK-8、细胞周期同步化、流式细胞术、基因芯片等方法研究Tmub1对细胞周期的影响，构建截断体来研究Tmub1泛素样及跨膜结构域在细胞增殖和影响securin泛素化的机制。结果：1、Tmub1与securin存在相互作用；2、Tmub1是细胞周期和肝细胞增殖的负向调控因子；3、Tmub1通过与泛素竞争结合E2作用位点从而负向调控有丝分裂的进程；4、Tmub1是一个细胞周期相关蛋白，可在多个细胞周期与cyclin A2相互作用起到对细胞周期的调控作用。结论：Tmub1通过间接的占位作用抑制securin泛素化，从而负向调控肝细胞增殖和肝再生。