【作者】 贺钰磊；

【导师】 曹励之；

【作者基本信息】 中南大学 ， 儿科学， 2009， 博士

【摘要】 WASP家族富含脯氨酸同源蛋白1(WASP-family verprolinhomologous protein 1,WAVE1)为近年来发现的新型肌动蛋白调节蛋白,在肌动蛋白聚合和细胞骨架重排中发挥重要作用。近年研究发现WAVE1在人类血液系统肿瘤细胞株中高表达。我们前期研究中发现WAVE1在儿童急性白血病患儿骨髓单个核细胞中高表达,并且与白血病病程相关,但其参与白血病致病的机制尚不清楚。已有研究发现了WAVE蛋白家族参与了实体肿瘤细胞的侵袭转移,但WAVE1是否参与与白血病细胞侵袭转移尚不清楚。本研究首先利用包含84个重要肿瘤通路基因的SuperArray PCR基因芯片研究了转染pcDNA3.1-WAVE1质粒后高表达WAVE1的白血病K562细胞系,发现转染后基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、整合素β3(Integrin,beta 3)等与肿瘤细胞侵袭转移的相关基因表达明显上调。提示WAVE1可能参与了肿瘤侵袭转移。接着以表达上调最明显的MMP-2为研究对象发现:转染pcDNA3.1-WAVE1 24h及48h后K562细胞MMP-2 mRNA和蛋白表达水平明显上调,而转染WAVE1 siRNA 24h及48h后K562细胞MMP-2 mRNA及蛋白水平明显下调。说明WAVE1可能参与了MMP-2表达的调控。进一步研究发现WAVE1促进了K562细胞丝裂原激活的的蛋白激酶(MAPK)通路的细胞外信号调节激酶(ERK1/2)的磷酸化,WAVE1可能通过这一途径影响MMP-2的表达。通过免疫荧光技术发现WAVE1与MMP-2在K562细胞的细胞膜上存在共定位。最后利用transwell方法检测了WAVE1表达对K562细胞侵袭能力的影响,发现与对照组K562细胞相比转染pcDNA3.1-WAVE1后细胞侵袭能力增强,而转染WAVE1 siRNA干扰WAVE1表达后K562细胞侵袭能力减弱。综上,本研究证明了WAVE1参与了K562白血病细胞侵袭的过程,其机制可能和调控MMP-2的表达并与MMP-2协同作用促进了白血病细胞的侵袭转移有关。WAVE1可以作为抗白血病细胞侵袭的新靶点。更多还原

【Abstract】 WASP-family verprolin-homologous protein 1(WAVE1)is a member of the actin regulatory protein family and play an important role in regulating actin’s polymerization and cytoskeleton’s reorganization.Re- cently,over-expression of WAVE1 in human blood cancer cell lines was reported.In our previous researches,high level of WAVE1 was found in mononuclear cells obtained from children’s bone marrow who suffered from acute leukaemia,but its role in leukaemia is still not clear.WAVE protein family was founded involved in invasion and migration of solid tumor cells.However,WAVE1 is involved in invasion and migration in leukemia cells is unclear.Super Array PCR genechip which contains 84 genes primers and expresses in many important cancer pathway was used in the study.Higher level of several genes including MMP-2,MMP-9 and Integrinβ3 were found in K562 leukaemia cells transfected with pcDNA3.1-WAVE1,those genes are believed involving in the process of leukemia’s invasion.WAVE1 may have some impact on tumor’s invasion.Expression of MMP-2 on mRNA and protein’s level was markedly increased by transfecting pcDNA3.1-WAVE1 in K562 cells,while MMP-2’s level is much lower in cells transfected with siRNA of WAVE1. Further study found that WAVE1 may promote phosphorylation of ERK1/2 in MAPK pathway through this way WAVE1 may affect the expresion of MMP-2.By using immunofluoresence,co-localization of WAVE1 and MMP-2 in cell membrane could been shown.To detect WAVE1’s effect on invasion ability of K562 cells, Transwell assay was performed.Invasion ability was enhanced in cells transfected with pcDNA3.1-WAVE1 while this ability was inhibited after using siRNA of WAVE1.In conclusion,WAVE1 may involve in migration and invasion of K562 cells by regulating the expression of MMP-2 and also by coordinating with MMP-2.WAVE1 could be a novel target to inhibit leukemia cells’ invasion in the future.更多还原