【摘要】 目的:构建稳定表达外源c-myc基因的人单核细胞白血病细胞株U937,并初步分析其特性。方法:首先构建重组质粒MSCV-c-myc-IRES-GFP(MMIG)载体,分别用MMIG及空载体MSCV-IRES-GFP(MIG)包装病毒,并感染U937细胞,用流式细胞术分选绿色荧光细胞,获得U937/GFP和U937/MYC细胞,用荧光显微镜及流式细胞术检测GFP阳性率,用Western blotting测定细胞中c-Myc、survivin、X连锁凋亡抑制蛋白(XIAP)和Bcl-2的蛋白表达水平,流式细胞术检测U937/GFP和U937/MYC细胞周期,并用MTT法测定U937/GFP和U937/MYC细胞的生长情况,克隆形成实验检测克隆形成能力。结果:荧光显微镜观察,MIG和MMIG病毒感染后,2种细胞均表达绿色荧光蛋白;流式细胞术结果显示,MIG病毒感染的细胞荧光率为26.0%,MMIG病毒感染的细胞荧光率为27.7%;Western blotting的结果显示,c-Myc蛋白在MMIG病毒感染的细胞中表达水平升高。流式细胞术分选后,荧光显微镜观察可见绿色荧光蛋白表达明显增多,U937/GFP和U937/MYC细胞绿色荧光蛋白表达率分别达98.7%和93.7%。U937/MYC细胞中c-Myc蛋白表达较U937/GFP细胞显著升高,c-Myc蛋白下游的survivin表达增多,而凋亡相关蛋白XIAP及Bcl-2的表达则没有明显变化。细胞周期检测显示,U937/MYC细胞处于S期的细胞数增多。MTT实验结果显示,U937/MYC细胞的生长速率较U937/GFP细胞增快。U937/MYC细胞的克隆形成能力较U937/GFP细胞强。结论:成功构建了c-myc基因高表达的U937稳定细胞株U937/MYC。在U937/MYC细胞中,c-Myc及其下游的survivin表达明显上调,处于细胞周期S期的细胞数增多、细胞生长加快、克隆形成能力增强,提示c-Myc可能通过增强自我更新能力、加快细胞周期、促进相关抗凋亡蛋白表达从而提高细胞的存活率。更多还原

【Abstract】 AIM: To establish a human monocytic leukemia cell line U937 stably expressing c-myc gene and to investigate the biological characteristics of this cell line. METHODS: The recombinant plasmid MSCV-c-myc-IRES-GFP( MMIG) was constructed. MMIG and MSCV-IRES-GFP( MIG) were used to package the viruses for infecting U937 cells.Fluorescence-activated cell sorter( FACS) was used for sorting U937 / GFP and U937 / MYC cells. The GFP-positive cells were detected by fluorescence microscopy and FACS. The protein expression of c-Myc,survivin,X-linked inhibitor of apoptosis protein( XIAP) and Bcl-2 was detected by Western blotting. Cell proliferation was evaluated by MTT assay. Propidium iodide( PI) staining was used to determine the cell cycle distribution. Self-renewal ability was observed by colonyforming assay. RESULTS: The GFP expression in the cells infected with MIG or MMIG virus was observed under fluorescence microscope. The green fluorescent rate of the cells infected with MIG was 26. 0%,while that of the cells infected with MMIG was 27. 7%. The protein expression of c-Myc in MMIG-infected U937 cells was higher than that in MIG-infected cells. After sorting,the green fluorescent rates of U937 / GFP and U937 / MYC cells reached 98. 7% and 93. 7%,respectively. The protein expression of c-Myc in U937 / MYC cells was higher than that in U937 / GFP cells. In addition,survivin,a downstream protein of c-Myc,was up-regulated,while the protein expression of XIAP and Bcl-2 remained unchanged. Cell cycle analysis showed that the percentage of the cells in S phase increased in U937 / MYC cells. Moreover,the proliferation and colony-forming ability of U937 / MYC cells were also enhanced. CONCLUSION: U937 / MYC cell line stably expressing c-myc gene was successfully established. c-Myc may increase cell viability via enhancing the expression of anti-apoptotic protein survivin,the cell cycle transition and the self-renewal ability.更多还原