核干细胞因子特异性短发夹状RNA的体内外干扰作用研究

发布时间：2018-08-01 14:49

【摘要】：目的：核干细胞因子(nucleostemin，NS)是新发现的一个蛋白，位于干细胞和肿瘤细胞的核仁上。当细胞处于早期多潜能状态增殖时NS呈高丰度表达，细胞开始分化时它的表达在细胞退出细胞周期之前迅速显著下调。它可能扮演了维持干细胞和肿瘤细胞自我更新的角色，NS缺失或过度表达均可使干细胞和肿瘤细胞的增殖减弱。在造血系统中，NS被发现表达于有c-kit~+/lin~-标志的骨髓干细胞中，但在成熟粒细胞与淋巴细胞中则没有。急性白血病是造血干细胞的恶性克隆性疾病，我们应用RT-PCR技术发现白血病细胞系HL-60和K562细胞中NS呈高表达。RNA干扰(RNA interference，RNAi)是基因转录后沉默作用(post-transcriptional gene silencing，PTGS)的重要机制之一，小干扰RNA(small interference RNA，siRNA)能高效特异地阻抑特定基因的表达。短发夹状RNA(short hairpin RNA，shRNA)在胞质内被核酸酶(Dicer)切割成siRNAs，发挥对靶基因的抑制作用。shRNA化学稳定性更高，又可在体外大量合成，易于筛选有效序列。我们在体外合成NS特异性短发夹状RNA并转染HL-60细胞，，通过观察细胞形态变化和测定NS抑制
[Abstract]:Objective: nuclear stem cell factor (NS) is a novel protein located on nucleoli of stem cells and tumor cells. The expression of NS was high when the cells were in the early multipotential state of proliferation, and the expression of NS was down-regulated rapidly and significantly before the cell withdrew from the cell cycle when the cells began to differentiate. It may play a role in maintaining the self-renewal of stem cells and tumor cells. The deletion or overexpression of NS can reduce the proliferation of stem cells and tumor cells. In the hematopoietic system, NS was found to be expressed in bone marrow stem cells with c-kit- / linlin-marker, but not in mature granulocytes and lymphocytes. Acute leukemia is a malignant clonal disease of hematopoietic stem cells. We found that the expression of RNA interference RNAi in leukemia cell lines HL-60 and K562 cells is one of the important mechanisms of post-transcriptional gene silencing. Small interfering RNA (small interference siRNAs can inhibit the expression of specific genes efficiently and specifically. Short hairpin RNA (short hairpin RNAs are cut into siRNAsby nuclease (Dicer) in the cytoplasm. The inhibitory effect of shRNA on target genes is more stable. ShRNA can be synthesized in vitro and is easy to screen out effective sequences. NS specific short hairpin RNA was synthesized and transfected into HL-60 cells in vitro. The changes of cell morphology and the inhibition of NS were observed.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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