HPO-X的分离纯化及分子生物学研究

发布时间：2018-05-07 14:18

本文选题：肝细胞生成素X + 肝脏再生　；参考：《中国人民解放军军事医学科学院》2005年博士论文

【摘要】：哺乳动物肝脏具有强大的再生能力,一般情况下,人的肝脏在受损后3天之内启动肝再生,2-3周后肝脏功能基本恢复,3-6个月后肝脏大小可恢复到与受损前一样。而成年哺乳动物肝细胞大部分长期处于G_0期,只有当肝脏在部分切除或受到损伤时,残余肝细胞才能进行快速的分裂,进而使残余肝组织代偿性增生。因此,作为一种器官、组织再生的理想模型,肝脏损伤及其再生的分子生物学机制一直是人们研究的热点课题之一。我国是一个肝病大国,病毒性肝炎、肝硬化与肝癌病人数在1.2亿左右,因而深入肝细胞损伤与再生的分子生物学机制研究,开发治疗肝损伤的药物,具有十分重要的社会意义。肝刺激物(hepatic stimulator substance,HSS)是一种能特异启动并促进肝细胞增殖的活性物质。近30年来,国内外学者围绕肝再生的机制.对动物源及人源的HSS作了大量深入的研究,对其组织来源、理化性质、分离纯化、生物学性能以及应用前景等都有了较深入的了解,但始终未能分离得到具有生物活性的HSS纯品,无法确定其分子结构,对其作用机理的研究也还有待阐明。我们实验室从上世纪80年代开始致力于肝刺激物的分离纯化研究,获得了一种相对分子量在(10-30)×10~3Da之间,能特异刺激肝源细胞增殖,对CCl_4引起的肝损伤具治疗作用的细胞组分,命名为人肝细胞生成素(hepatopoietin,HPO),并于1995年获得美国专利。但由于其纯度未能达到氨基酸序列测定的标准,所以始终未能获得其氨基酸序列,从而不能确定其真正的身份。在本研究中,我们采用DEAE-cellulose和Source15Q离子交换层析、聚丙烯酰胺凝胶电泳分离后割胶回收的方法从新生小牛肝脏中分离得到了三个具有体外促进肝癌细胞增殖的蛋白质,经多次富集后进行Q-TOF质谱测序,鉴定出其中之一是一个富亮氨酸的酸性核蛋白(Leucine-rich acidic nuclear protein,LANP),其同源的人源体是pp32。pp32是一个多功能的酸性核蛋白,在正常组织可自我更新的细胞和肿瘤组织中都有较高水平的表达。研究发现其在细胞的信号转导、蛋白质降解、细胞骨架动力学以及形态发生等过程中都参与作用,但体外促进肝细胞增殖方面目前没有报道,因此我们也将次蛋白命名为HPO-X。
[Abstract]:The mammalian liver has a strong regenerative ability. In general, the liver function of the human liver can be restored to the same size as before after the liver regeneration is started within 3 to 3 weeks after injury and the liver function recovers after 3 to 6 months. However, most of the adult mammalian hepatocytes are in the G _ S _ 0 phase for a long time. Only when the liver is partially resected or damaged can the residual liver cells divide rapidly and the residual liver tissue proliferate compensatively. Therefore, as an ideal model of organ and tissue regeneration, the molecular biological mechanism of liver injury and regeneration has been one of the hot topics. China is a large country with liver diseases, the number of patients with viral hepatitis, liver cirrhosis and liver cancer is about 120 million. Therefore, it is of great social significance to study the molecular biological mechanism of liver cell damage and regeneration and to develop drugs for the treatment of liver injury. Hepatic stimulator substance1 (HSS) is a kind of active substance which can specifically initiate and promote the proliferation of hepatocytes. In the past 30 years, scholars at home and abroad have focused on the mechanism of liver regeneration. In this paper, we have made a lot of in-depth studies on HSS from animal and human sources, and have a deep understanding of its tissue origin, physicochemical properties, separation and purification, biological properties and application prospects. However, the pure HSS with biological activity can not be separated and its molecular structure can not be determined, and the mechanism of its action is still to be elucidated. Our laboratory began to study the isolation and purification of liver irritants in the 1980s, and obtained a cell component with a relative molecular weight of 10-30 脳 10~3Da, which can specifically stimulate the proliferation of hepatogenic cells and has therapeutic effect on liver injury induced by CCl_4. Human hepatopoietin was named hepatopoietin HPOA and was patented in the United States in 1995. However, its purity can not meet the standard of amino acid sequence determination, so it can not obtain its amino acid sequence, so it can not determine its true identity. In this study, three proteins were isolated from newborn calf liver by DEAE-cellulose and Source15Q ion exchange chromatography and polyacrylamide gel electrophoresis. Q-TOF mass spectrometry sequencing showed that one of the leucine-rich acidic nucleoproteins Leucine-rich acidic nuclear protein was identified as a multifunctional acidic nucleoprotein. The homologous human body was pp32.pp32, which was a multifunctional acidic nucleoprotein. There is a high level of expression in self-renewing cells and tumor tissues in normal tissues. It has been found that HPO-X is involved in signal transduction, protein degradation, cytoskeleton dynamics and morphogenesis. However, there is no report on the promotion of hepatocyte proliferation in vitro, so we also named the secondary protein HPO-X.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

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