【作者】 裘捷；

【导师】 李载平； 赵慕钧；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 四跨膜蛋白是一个特殊的细胞膜糖蛋白家族,通常由4个高度疏水的保守跨膜区段,2个较短的胞内区,以及2个差异相对较大的胞外区构成。四跨膜蛋白参与细胞生长增殖、黏附、迁移和浸润等多种功能,是一个极其重要的蛋白质家族。TM4SF4蛋白(Transmembrane 4 superfamily member 4)是一个四跨膜蛋白超家族的成员,最初是作为一个在人小肠上皮细胞和肝细胞中表达的四跨膜糖蛋白被发现的,能够调控细胞密度相关的增殖抑制。我们实验室首先克隆了大鼠中的同源基因rat TM4SF4,并发现它在2/3肝切除后的再生肝中表达升高,推测可能与肝细胞再生相关。但是该蛋白在体内的具体功能和作用机制还不清楚。本论文的第一部分工作,我们采用四氯化碳大鼠急性肝损伤模型,研究了TM4SF4在四氯化碳急性肝损伤中作用。RT-PCR和Western blot检测表明,在四氯化碳急性肝损伤的过程中,TM4SF4的mRNA和蛋白表达都急剧升高。用尾静脉注射的方法,将正义的或反义的TM4SF4表达质粒注入大鼠体内,上调或下调肝脏内TM4SF4的表达;然后用血清ALT/AST活力、肝脏组织学损伤评定等指标来评价肝脏的损伤程度,并用荧光实时定量PCR和Western blot等方法来检测损伤相关基因表达情况的变化。结果表明,TM4SF4的过表达,会加强肝脏的损伤,并使血清ALT、AST活力的升高。而下调TM4SF4基因的表达,则会减轻肝脏的坏死程度,降低血清ALT、AST的活力。TM4SF4的表达上调,还会影响一些生长因子和受体的表达水平,如TNF-α、TNFR1和c-met等。此外,TUNEL检测表明,TM4SF4的表达上调,还能促进CCl4引起的肝细胞凋亡,并且改变一些促凋亡基因和抑凋亡基因的表达。因此,我们的实验表明大鼠TM4SF4基因在四氯化碳诱导的肝脏急性损伤中表达上升,并且在促进肝脏损伤中起重要的作用,其作用机制可能与TNF-α和HGF/c-met信号通路有关。本论文的第二部分工作,我们对TM4SF4蛋白在肝细胞癌中的表达和功能进行了研究。对临床肝癌和癌旁样品的Western blot和免疫组化的检测表明,TM4SF4蛋白在肝细胞癌中高表达,而在相应癌旁组织中的表达明显较低或检测不到。在肝癌细胞中导入TM4SF4表达质粒,TM4SF4基因的高表达能显著促进肝癌细胞的克隆形成能力和细胞增殖速度。采用RNA干扰技术抑制肝癌细胞BEL-7404中TM4SF4基因的表达后,能明显抑制细胞的生长和侵袭能力。免疫荧光共定位实验表明,TM4SF4定位于细胞膜上,并且能在EGF的刺激作用下进入到细胞质中。同时发现抑制TM4SF4的表达,能够使EGFR通路下游JAK激酶和STAT3的磷酸化程度减弱;过表达TM4SF4能够增强STAT3-诱导的启动子的转录活力。我们的实验表明,TM4SF4基因在肝细胞癌中表达上升,TM4SF4的过表达能够促进肝癌细胞的恶性增殖,并参与EGFR/STAT3信号通路的活化。我们的工作第一次对TM4SF4的功能进行了较深入地研究,由此发现了TM4SF4的异常高表达能促进肝脏的损伤和肝癌细胞的恶性增殖,并且第一次对TM4SF4的作用机理进行了初步的探讨,发现TM4SF4与TNF-α、HGF/c-Met和EGFR/STAT3信号通路有关。目前对于这个基因的功能和作用机制、以及表达调控等方面正在进行深入的研究。更多还原

【Abstract】 Transmembrane 4 superfamily is a group of hydrophobic proteins with four transmembrane domains and two extracellular loops, both with conserved residues. They are expressed in a wide variety of cell types and have functional roles in processes, such as cellular adhesion, migration, proliferation and tumour invasion. TM4SF4 (transmembrane 4 superfamily member 4, also known as intestine and liver tetraspan membrane protein, il-TMP) is a distant member of the transmembrane 4 superfamily that was originally identified as a tetraspan membrane glycoprotein produced in the human intestinal eptithelium and in non-dividing hepatocytes that can mediate the cell density-associated inhibition of proliferation. Our lab firstly cloned the rat homolog of TM4SF4 gene. Rat TM4SF4 was identified as a gene up-regulated in liver regeneration and supposed to be a proliferation-associated gene. However, the in vivo functions of TM4SF4 and the possible molecular mechanism for TM4SF4 action are largely unknown.Firstly, we investigated the in vivo function of TM4SF4 in a rat carbon tetrachloride (CCl4)-mediated liver injury model. Expression of TM4SF4 was analyzed by RT-PCR and Western blot in normal and CCl4-injured rats. Our study shows that TM4SF4 gene is overexpressed in acutely injured liver. Overexpression or reduced expression of TM4SF4 in the liver was achieved by injection of sense or antisense TM4SF4 expression plasmids. Assessment of liver injury (histology, serum ALT and AST levels), apoptosis by TUNEL assay were performed. TM4SF4 overexpression is found to be associated with more elevated ALT/AST, increased necrosis, and enhanced apoptosis. Decreased TM4SF4 gene expression showed minimal liver necrosis and depressed ALT/AST levels. Expression of injury-related genes was analyzed by quantitative real-time PCR. Increased expression of TM4SF4 affected the expression levels of growth factors and receptors, such as TNF-α, TNFR1 and c-met. Furthermore, pro-apoptotic and anti-apoptotic gene expression was altered after TM4SF4 administration. Our findings suggest that TM4SF4 plays an important role in accelerating CCl4 liver injury, which may be mediated by the TNF-αand HGF/c-met signaling pathways.Secondly, we investigated the relation between TM4SF4 expression and hepatocellular carcinoma. Western blot and immunohistological analysis showed that the TM4SF4 expression was most abundant in HCC tissues and significantly reduced or undetected in corresponding noncancerous tissues. After introduction of the sense and antisense cDNA of TM4SF4 into HCC cell line QGY-7701 and BEL-7404, we observed that the overexpression of TM4SF4 enhanced both colony formation and cell proliferation in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of TM4SF4 gene in BEL-7404 HCC cell lines resulted in the cell growth greatly inhibited. Further immunofluresence analysis explored that TM4SF4 was localized on the cellular membrane and could translocate from membrane to cytoplasma with EGFR under the induction of EGF. Moreover, reduction of the TM4SF4 gene expression could also inhibit the phosphorylation of JAK and STAT3. Overexpression of TM4SF4 gene enhances transcriptional activity of STAT3-regulated promoter, which is inhibited by an inhibitor of Janus tyrosine kinase activity. Therefore, TM4SF4 might be involved in EGFR/STAT3 signaling pathway.In conclusion, TM4SF4 gene is up-regulated in acute liver injury and hepatocellular carcinoma. Overexpression of TM4SF4 accelerates CCl4-induced acute liver injury, which might be mediated by the TNF-αand HGF/c-met signaling pathways. In addition, overexpressing TM4SF4 could enhance cell proliferation and tumor invasion in HCC, which might be associated with EGFR/JAK/STAT3 pathway. The further functional analysis and the transcriptional regulation of this gene are being performed now.更多还原