CAJ | 학술논문

背景与目的:CCAAT-增强子结合蛋白(CCAAT-enhancer binding proteinε,C/EBPε)是一种在髓系细胞中特异性表达的核内转录因子,可能是髓系细胞分化过程中重要的调控因子,并且参与了一系列髓系细胞特异性基因的转录激活.本研究拟探讨C/EBPε的细胞特异性表达,研究过量表达C/EBPε对人粒-单核性白血病细胞U-937中c-Myc表达、细胞周期及细胞分化的影响.方法:应用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RTPCR)检测五种不同类型的造血系细胞中C/EBPε的表达水平.根据RT-PCR的结果,选择人粒-单核性白血病细胞U-937作为过量表达C/EBPε的对象.电穿孔转染C/EBPε表达质粒pcDNA3.1-C/EBPε,G418筛选稳定表达细胞并命名为U-937-C/EBPε32.RT-PCR与Western blot检测转染细胞内C/EBPε与c-Myc的表达水平,流式细胞仪检测过量表达C/EBPε对U-937细胞周期及细胞分化的影响.结果:过量表达C/EBPε可以显著增强转染U-937细胞表面粒细胞特异性表面抗原CD11b的表达,U-937-C/EBPε32细胞表面CD11b的阳性率达92.56%,而在U937与U-937-pcDNA3.1细胞表面分别为77.46%与74.81%,但c-Myc的表达与细胞周期分布未受影响.结论:C/EBPε可能是髓系细胞向粒细胞分化过程中非常重要的转录调控因子.
배경여목적:CCAAT-증강자결합단백(CCAAT-enhancer binding proteinε,C/EBPε)시일충재수계세포중특이성표체적핵내전록인자,가능시수계세포분화과정중중요적조공인자,병차삼여료일계렬수계세포특이성기인적전록격활.본연구의탐토C/EBPε적세포특이성표체,연구과량표체C/EBPε대인립-단핵성백혈병세포U-937중c-Myc표체、세포주기급세포분화적영향.방법:응용역전록-취합매련반응(reverse transcription-polymerase chain reaction,RTPCR)검측오충불동류형적조혈계세포중C/EBPε적표체수평.근거RT-PCR적결과,선택인립-단핵성백혈병세포U-937작위과량표체C/EBPε적대상.전천공전염C/EBPε표체질립pcDNA3.1-C/EBPε,G418사선은정표체세포병명명위U-937-C/EBPε32.RT-PCR여Western blot검측전염세포내C/EBPε여c-Myc적표체수평,류식세포의검측과량표체C/EBPε대U-937세포주기급세포분화적영향.결과:과량표체C/EBPε가이현저증강전염U-937세포표면립세포특이성표면항원CD11b적표체,U-937-C/EBPε32세포표면CD11b적양성솔체92.56%,이재U937여U-937-pcDNA3.1세포표면분별위77.46%여74.81%,단c-Myc적표체여세포주기분포미수영향.결론:C/EBPε가능시수계세포향립세포분화과정중비상중요적전록조공인자.
BACKGROUND & OBJECTIVE: CCAAT-enhancer binding protein ε (C/EBPε) is a kind of nuclear transcriptional factor expressed predominantly in myeloid cells, and may be a critical regulator of myeloid differentiation, which can activate the transcription of a subset of myeloidspecific genes. This study was to detect the cell-specific expression of C/EBPε, and to investigate the effect of C/EBPε overexpression on c-Myc expression, cell cycle distribution, and cell differentiation of human myelomonocytic leukemia cell line U-937. METHODS: The expression of C/EBPε in 5 hematopoietic cell lines (U-937, NB4, HL-60, K-562 and Jurkat T cell lines) was tested by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). According to the RT-PCR results, human myelomonocytic leukemia cell line U-937 was selected and transfected with pcDNA3.1-C/EBPε32 expression vector by electroperforation, and screened by G418 to yield positive clones which were termed U-937-C/EBPε32. The expression of C/EBPε and c-Myc in U937-C/EBPε32 cells was detected by RT-PCR and Western blot; cell cycle and differentiation of U937-C/EBPε32 cells was analyzed by flow cytometry. RESULTS: C/EBPε overexpression obviously increased the expression of CD11b (a ceil surface marker of granulocyte maturity). The positive rate of CD11b was significantly higher in U-937-C/EBPε32 cells than in U-937 and U-937-pcDNA3.1 cells(92.56% vs.77.46% and 74.81% ), while c-Myc expression and cell cycle had no changes. CONCLUSION: C/EBPε might be an essential transcriptional regulator for U-937 cells differentiating into granulocytes.