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目的:确定控制脐血造血干/祖细胞增殖的细胞周期蛋白D(cyclinD)关键亚型,体外观察再生障碍性贫血(再障)患者血清对脐血造血干/祖细胞cyclinD关键亚型表达的影响,以探讨再障的发病机制。方法:用免疫磁珠阳性分选的方法分离脐带血CD34+细胞,用RT-PCR和Westernblotting方法检测其cyclinD各亚型的表达以筛选其关键亚型;用甲基纤维素半固体培养方法,观察再障血清对脐血CD34+细胞粒单系-集落形成单位(CFU-GM)形成的影响;再用半定量RT-PCR和Westernblotting方法检测再障血清对脐血CD34+细胞cyclinD关键亚型表达的影响。结果:RT-PCR和Westernblotting结果显示:控制脐血CD34+细胞增殖的cyclinD关键亚型为cyclinD3;再障血清可以显著抑制脐血CFU-GM的形成,并分别可以从mRNA和蛋白质水平抑制cyclinD3亚型的表达。结论:CyclinD3是控制脐血CD34+细胞增殖的关键cyclinD亚型,再障血清可以抑制其表达,这可能是再障造血抑制的一种机制。
OBJECTIVE: To determine the key subtypes of cyclinD that control the proliferation of hematopoietic stem / progenitor cells in vitro and to observe the expression of cyclinD in umbilical cord blood hematopoietic stem / progenitor cells from the serum of patients with aplastic anemia (aplastic anemia) in vitro Influence, to explore the pathogenesis of aplastic anemia. Methods: Cord blood CD34 + cells were isolated by immunomagnetic beads positive method. The expression of cyclinD subtypes was detected by RT-PCR and Westernblotting to screen their key subtypes. Methylcellulose semi-solid culture The effect of aplastic anemia on the formation of CD34 + monocyte-colony forming unit (CFU-GM) in umbilical cord blood was studied. The effect of aplastic anemia on cyclinD expression in cord blood CD34 + cells was detected by semi-quantitative RT-PCR and Western blotting . Results: RT-PCR and Western blotting showed that the key cyclinD subtype that controls the proliferation of cord blood CD34 + cells was cyclinD3. Aplastic anemia could significantly inhibit the formation of CFU-GM in umbilical cord blood and inhibit the expression of cyclinD3 subtype from mRNA and protein level expression. Conclusion: CyclinD3 is a key cyclinD subtype that controls the proliferation of cord blood CD34 + cells. Aplastic anemia can inhibit the expression of CyclinD3, which may be a mechanism of aplastic anemia.