绵羊α-TTP转运α-生育酚过程关键因子的功能及其调控机制研究

α-tocopherol transfer protein (α-TTP) is an important factor, which is responsible for the transport of α-tocopherol in mammals. However, only a few reports about α-TTP were found by now and concentrated on human and mice, the research field in livestock is still absent. We have cloned the full-length cDNA sequence of the ovine α-TTP gene, and found this protein is mainly expressed in liver and located in liver cytoplasm. The interacting proteins and regulatory factors were then identified, including myosin and small GTPase, which were believed to involve in the transport process. Based on our previous results and the latest reports in this field, the present research aims to construct the primary sheep liver cells and transfected with α-TTP gene. The locations of α-TTP, myosin and small GTPase will be detected. The expressions of myosin and small GTPase and the content of phosphatidylinosital biphosphate will be determined in response to appropriate supplementation of α-tocopherol. The technology of RNA interference and treatment of inhibitor will also be used to investigate the function of myosin, small GTPase and phosphatidylinosital biphosphate during the transport process. Besides, the omics technology will be applied to identify other possible modulating factors. This is a novel research in the relative field, which will fill the blanks on ovine α-TTP. The results will also provide references for the transport process of α-tocopherol by α-TTP in liver, as well as its molecular mechanisms.

α-生育酚转运蛋白（α-TTP）是哺乳动物转运α-生育酚的重要因子。目前有关研究较少且集中于人和鼠，畜禽上未见报道。本团队前期成功克隆绵羊α-TTP基因，发现其主要在肝脏中表达，且定位于肝细胞质中；筛选出参与转运α-生育酚过程的功能因子——肌球蛋白和小GTP酶。在此基础上，结合最新报道，本研究拟构建绵羊肝细胞模型并转染α-TTP基因，开展α-TTP与肌球蛋白和小GTP酶肝细胞内定位研究；测定α-生育酚最适浓度下肌球蛋白与小GTP酶的表达水平及磷脂酰肌醇二磷酸含量；采用RNA干扰或抑制剂处理对其在转运过程中的功能进行研究；并运用组学技术挖掘其他可能的调控因子。本课题选题新颖，研究内容处于领域前沿，研究结果对阐明α-生育酚在机体内的转运及机理，完善α-TTP的生物学功能，填补绵羊相关研究的空白，具有重要的科学意义和参考价值。

α-TTP是哺乳动物转运α-生育酚的重要因子，目前研究较少且只集中于人和鼠。本团队前期成功克隆羊α-TTP基因，发现其主要在肝脏中表达，且定位于肝细胞质中，筛选出参与转运α-生育酚过程的功能因子—肌球蛋白和小GTP酶。在此基础上，本项目进行了24个系列试验的6项研究，完成绵羊肝实质细胞分离、鉴定、原代培养；确定体外培养肝细胞α-生育酚处理的最佳条件为100 μmol/L，24 h；筛选出Rab小GTP酶、肌球蛋白和热休克蛋白三类候选α-TTP互作蛋白；发现Rab8a、Rab14、Rab21、MYO1D、TPM3、CD71、Hsp27、Hsp90ab1、SNX3、SNX5、SNX17和SNX27蛋白可能与α-TTP共定位，Rab8a和Hsp90ab1与α-TTP存在蛋白互作；Rab14、Rab21、Hsp27和SNX3与α-TTP未出现互作，可能不具有调控α-生育酚转运功能；Rab8a、Hsp90ab1和Myo1d参与了维生素E转运过程；筛选出Rab8a、Vps26a、Vps35、SNX3、SNX17、SNX27等受VE影响而上调的关联基因；证明敲降SNX5、SNX17和SNX27能影响α-TTP转运α-生育酚过程，推测retriever-SNX17、retromer-SNX27和ESCPE1（含SNX1、SNX2和SNX5）蛋白复合物可能是α-TTP携带α-生育酚从循环内体或晚期内体/溶酶体向质膜运输的重要调节器和载体。本项目深入研究α-生育酚胞内转运过程，阐明α-生育酚在机体内转运和部分作用机理，对于完善α-TTP生物学功能，填补相关研究空白，具有重要的科学意义和参考价值。