p27基因下调对人骨髓和脐血造血祖细胞体外活性的影响  

The effect of down regulating of p27 on the activity of hematopoietic progenitor cells derived from umbilical cord blood and bone marrow ex vivo

作　　者：李莉[1] 李先兴[1] 程涛 

机构地区：[1]第二军医大学长征医院实验诊断科,上海200003 [2]Stem Cell Biology,University of Pittsburgh Cancer Institute

出　　处：《中华血液学杂志》2005年第2期90-93,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(30371360)

摘　　要：目的比较干扰p27基因表达对骨髓和脐血来源造血祖细胞增殖和造血潜能的影响,并探讨其相关机制。方法以含p27全长反义cDNA逆转录病毒感染经流式细胞仪分选的人骨髓与磁珠分离获得的人脐血来源的CD34+细胞,在含混合生长因子条件下体外培养不同时间,分别观察细胞生长曲线,检测细胞周期,确定细胞增殖能力;半固体培养计数集落形成,确定其造血能力;Westernblot检测p27与CDK2蛋白表达,明确基因转染效果并探讨p27反义cDNA促进细胞扩增的作用途径。以含p27全长正义cDNA和仅含绿色荧光蛋白(GFP)基因的病毒感染细胞为对照。结果与GFP和p27正义cDNA比较,p27反义cDNA对脐血造血祖细胞生长有明显促进作用(P<0.01),至培养第9天p27反义cDNA、p27正义cDNA和GFP组细胞数分别增加了(197.3±47.7)倍、(12.7±8.1)倍和(41.8±30.6)倍;骨髓造血祖细胞数增加不明显,至培养第9天3组细胞数分别增加了(36.0±22.3)倍、(8.7±6.8)倍和(14.1±10.4)倍。p27反义cDNA主要促进脐血和骨髓造血祖细胞S期增加,p27反义cDNA、p27正义cDNA和GFP组脐血造血祖细胞S期细胞百分比为(17.0±4.8)%,(2.0±0.8)%和(4.1±1.8)%;骨髓造血祖细胞则为(8.4±4.4)%,(1.0±0.7)%和(3.8±1.4)%。p27反义cDNA对骨髓和脐血的集落形成能力促进作用高于GFP组(P<0.Objective To compare p27 interfering effect on the proliferation and hematopoietic potential of hematopoietic progenitor cells(HPC)derived from human bone marrow(BM)and umbilical cord blood(UCB ), and investigate the related mechanism. Methods CD34+ cells sorted from human BM by flow cytometry and isolated from UCB by a magnetic isolation system were infected with retrovirus-p27 antisence cDNA(p27AS) and cultured with cocktail-cytokines. The cell proliferation capacities were detected by cell growth curve and DNA content analysis, and the hematopoietic potential by colony formation assay. The protein expression of p27 and CDK2 were measured with Western blot. CD34+ cells infected with retrovirus-p27 sense cDNA(p27SE) and virus-green fluorescence protein(GFP) were used as the control. Results Comparing with groups of GFP and p27SE, p27AS showed to accelerate the expansion of UCB HPC significantly (P<~0.01 ), the cell number of p27AS, p27SE and GFP increased by 197.3±47.7-, 12.7±8.1-and 41.8±~30.6 -fold respectively by the end of the 9-day culture, the BM HPC increased by 36.0±22.3-, 8.7±6.8-and 14.1±10.4-fold respectively in the same time as UCB HPC. Cell cycle analysis showed p27AS mainly promoted S phase of BM and UCB HPC. S phase cell percentages of UCB HPC infected with p27AS, p27SE and GFP were (17.0±4.8)%,(2.0±0.8)% and (4.1±1.8)% and that of BM HPC were (8.4±4.4)%,(1.0±0.7)% and (3.8±1.4)% respectively. The yields of colony formation of p27AS for BM and UCB was higher than that for GFP (P<0.05). Western blot demonstrated the expression of p27 reduced in the p27AS group, while CDK2 increased in the group. The virus infection rate, cell growth rate and colony forming yields of BM HPC was inferior to that of UCB HPC. Conclusions p27 gene interfering could promote HPC proliferation, hematopoietic potential and the response to stimulations of UCB HPC is higher than that of BM HPC ex vivo. It seems that cell cycle controlled by p27 was related to CDK2.

关 键 词：反义CDNA 脐血 骨髓造血祖细胞 P27基因 CDK2 集落形成 人骨髓 绿色荧光蛋白(GFP) 体外活性 细胞扩增 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]