bmi-1原癌基因在不同来源CD34^＋细胞的表达

Expression of bmi-1 in CD34～+cells from different sources

目的:Bmi-1是一种属于PcG家族的原癌基因,它直接参与细胞生长、增殖的调节,是成体干细胞和白血病干细胞自我更新所必需的。检测原癌基因bmi-1在不同来源CD34+细胞中的表达,探讨其与细胞增殖的关系。方法:实验于2007-03/11在广州医学院生化教研室完成。①实验材料:白血病细胞株SHI-1由苏州大学附属第一医院、江苏省血液病研究所薛永权教授惠赠;脐血由广州医学院附属广州市第一人民医院妇产科提供,实验经产妇知情同意,并经医院伦理委员会批准;外周血采自健康成年自愿捐献者。②实验方法:以CD34免疫磁珠标记急性单核细胞白血病细胞株SHI-1,上柱分选CD34+细胞,脐血单个核细胞以同样的方法标记CD34免疫磁珠标记和分选CD34+细胞,应用流式细胞仪分析分选后CD34+的富集度;将分选前后的CD34+、CD34-SHI-1细胞以相同的密度接种于24孔板,分不同的时间点计数细胞,并绘制生长曲线。选用淋巴细胞分离液分离外周血单个核细胞。以正常脐带血和外周单个核细胞为对照,采用半定量RT-PCR法检测bmi-1基因在不同来源CD34+细胞中的表达。结果:①SHI-1细胞分选前后标记CD34-PE、CD38-FITC流式分析显示,分选后CD34+细胞的富集度为99%以上。②SHI-1-CD34+细胞呈缓慢生长趋势,不同于分选前细胞的指数生长状态。③RT-PCR检测显示,bmi-1mRNA在SHI-1-CD34+细胞中的表达高于其在脐血-CD34+与外周血单个核细胞中的表达(P<0.05)。bmi-1mRNA在SHI-1、SHI-1-CD34+、SHI-1-CD34-细胞中表达差异不显著,在脐血-CD34-细胞中弱表达或不表达。结论:bmi-1基因在SHI细胞株CD34+与CD34-细胞中均高表达,说明其与血液细胞的高增殖能力密切相关。

AIM： Bmi-I is a proto-oncogene belonging to the Polycomb group （PcG）. It is directly involved in the regulation of growth and proliferation of cells, and necessary for the self-renewal of adult and leukemia stem ceils. The present study aimed to investigate the expression of bmi- I in CD34^＋ cells from different sources of cells, and to explore the relation of bmi- I and proliferation of cells. METHODS： The experiment was performed at the Department of Biochemistry, Guangzhou Medical College from March to November 2007. Leukemia cell strain SHI-I was offered by Professor Xue from the First Affdiated Hospital of Soochow University, Institute of Hematology of Jiangsu Province. Umbilical cord blood （UCB/ was provided by the Department of Gynecology and Obstetrics of Guangzhou First People＇s Hospital of Guangzhou Medical College. The informed consents were obtained from puerperas and the experiment was approved by Hospital Ethics Committee. The peripheral blood was offered by the healthy adult donors. After CD34^＋ cells were isolated by Midi MACS from the SHI-I cells and UCB, the enrichment of CD34^＋ was assayed by flow cytometry. The proliferation of SHI-I, SHI-I-CD34^＋, SHI-I-CD34^- cells were observed by counting in different time after incubating in a 24-well plate. The peripheral blood mononuclear cells （PBMNC） were isolated from the healthy donors by lymphocyte isolation. The expression of bmi-I mRNA in above-mentionaed cells was measured by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）, normal UCB and PBMNC as controls. RESULTS： SHI-I cells before and alter sorting were assayed by flow cytometry marked CD34PE and CD38F1TC. The results showed the enrichment of SHI-CD34^＋ cells was up to 99%. SHI-1-CD34^＋ cells proliferated slowly, different from SHI-I cells before sorting growing exponentially. RT-PCR demonstrated that bmi-I mRNA expressions of SHI-I-CD34^＋ cells were remarkably higher than normal UCB- CD34^＋ and peripheral blood mononuclear ceils （P 〈 0.05）. The difference was not significant from SHI-1 and SHI-CD34^- cells, bmi-1 mRNA expression was very low or negative in UCB-CD34^- cells. CONCLUSION： Bmi-I highly expresses in both SHI-I-CD34^＋ and SHI-1-CD34^- cells, closely correlating with the proliferation of blood cells.