【作者】 肖鹏；

【导师】 马小京；

【作者基本信息】 上海交通大学 ， 生物学， 2015， 硕士

【摘要】 高尔基体磷酸化蛋白2(Golgi phosphoprotein 2,GOLPH2)是新近发现的一种高尔基体驻留蛋白,主要表达在上皮组织中。正常情况下GOLPH2锚定在高尔基体膜上,可能参与蛋白质翻译后修饰等过程,但是病理情况下GOLPH2会被分泌出细胞。有研究发现,在病毒性肝炎、肝硬化、肝细胞癌患者体内,GOLPH2在组织和血清中的表达量会显著上升,因此可以作为肝细胞癌早期检测的血清标记物,并且其特异性和灵敏性优于传统的肝细胞癌生物标记物甲胎蛋白。此外,最近的临床研究表明前列腺癌、肺癌、阿尔兹海默病等疾病患者体内,GOLPH2的表达量也会上升,显示其可能与多种疾病的发生或发展有广泛的关系。IL-12在诱导Th0细胞向Th1细胞分化的过程中起到了关键作用,而Th1细胞在机体抵抗胞内病原体感染以及抗肿瘤的细胞免疫中有重要作用。由于我们在体外实验中发现GOLPH2可以抑制IL-12两个亚基p35和p40在巨噬细胞中的转录,因此我们推测患者体内表达量上调的GOLPH2可以通过抑制IL-12间接地促进疾病的发生。为进一步研究GOLPH2的功能,我们通过TALEN技术构建出GOLPH2敲除小鼠,发现与野生型小鼠相比,GOLPH2敲除小鼠在体型外观、行为方式、血细胞组成、脾细胞组成及其他常见生理指标等方面没有显著区别。由于内毒素休克模型常用来研究机体的急性免疫反应并且能引起机体产生大量的IL-12,因此我们给小鼠腹腔注射LPS建立该模型并检测血清中主要免疫细胞因子的分泌情况。我们发现在注射LPS之后,GOLPH2敲除小鼠血清IL-12含量比野生型小鼠更高,这与我们在细胞水平的实验结果一致。为进一步了解GOLPH2在免疫学中可能起到的作用,我们运用RNA测序技术比较了野生型小鼠和GOLPH2敲除小鼠激活的骨髓来源树突状细胞的mRNA表达谱。测序结果的生物信息学分析显示,GOLPH2可能参与细胞外基质、细胞骨架蛋白等结构成分的调控。综上所述,本研究提供了GOLPH2基因敲除小鼠,并初步探讨了GOLPH2在调节免疫反应中可能起到的作用,为进一步研究其功能提供了一定的理论基础,也为GOLPH2在人类疾病治疗过程中成为药物研发的靶点提供了一定的理论依据。更多还原

【Abstract】 Golgi phosphoprotein 2(GOLPH2) is a Golgi-residing protein mainly expressed in epithelial tissues. In normal conditions, GOLPH2 is anchored on the Golgi membrane, and is speculated to be involved in post-translational modification of other proteins. However, GOLPH2 can be secreted outside cells under certain not well defined pathological conditions. Some studies found that GOLPH2 were up-regulated significantly in viral hepatitis, cirrhosis and in patients of hepatocellular carcinoma(HCC), both in the serum and in tissues. Thus, GOLPH2 levels could be a serum marker for early detection of HCC with potentially greater sensitivity and specificity than α-fetoprotein(AFP), a traditional biomarker for HCC for nearly half a century. Moreover, recent clinical studies show that in patients of prostate cancer, lung cancer and Alzheimer’s disease, GOLPH2 expression is also up-regulated, suggesting that it may play a broad role in the development of diseases.We first found that GOLPH2 could repress the transcription of two subunit genes of interleukin-12(IL-12), p35 and p40. IL-12 plays a key role in promoting the differentiation of na?ve T helper(Th) cells into Th1 cells, which are crucial for cell-mediated immunity against intracellular pathogens and cancer. Therefore, we hypothesize that the up-regulated GOLPH2 could promote the development of tumorigenesis indirectly through the inhibition of IL-12.In order to investigate its functions further, we constructed a GOLPH2 knockout(KO) mouse strain by the TALEN technology. GOLPH2 KO mice displayed little difference from WT mice in gross appearance, behavior, blood cell account, physiological index and spleen cell composition.Because endotoxin-induced shock model has been widely used to study the acute inflammatory response, we constructed such a model by intraperitoneal injection of LPS and checked the concentration of major immune cytokines in the serum. We found higher levels of serum IL-12 in GOLPH2 KO mice compared with WT controls, this is in accordance with the experiment results in vitro.To assess the role of GOLPH2 in the immune activities of dendritic cells(DCs), we compared the mRNA expression profiles of LPS-activated WT and KO DCs derived from the bone marrow by next generation RNA-sequencing. Bioinformatic analyses of the sequencing data suggest that GOLPH2 may be involved in the regulation of extracellular matrix, cytoskeleton or other structural components.Our studies begin to reveal the potential role of GOLPH2 in the hematopoietic system and non-hematopoietic tissues, also lay the foundation for further investigation of GOLPH2’s multifaceted activities in normal and pathogenic conditions.更多还原