【作者】 刘卫红；

【导师】 孟秀香；

【作者基本信息】 大连医科大学 ， 临床检验诊断学， 2005， 硕士

【摘要】 目的 白血病是一种严重威胁人类健康的恶性肿瘤。抗白血病疗法一般旨在消灭处于增殖期的细胞,但像正常及白血病造血干细胞这样较少处于增殖状态的细胞便能抵抗这种治疗,所以,白血病造血干细胞特异基因靶点的识别,特别是决定其自我更新基因靶点的识别,便有可能把白血病阻止在其源头,为白血病治疗提供新的手段。Bmi-1对于正常和白血病造血干细胞的自我更新至关重要,白血病造血干细胞或祖细胞如果缺乏 Bmi-1 会产生明显的增殖障碍,这使得 Bmi-1 基因可能成为治疗白血病的靶点。本研究通过构建反义 Bmi-1 的真核表达载体并转染 K562 细胞,目的在于使其产生的反义 mRNA 封闭 K562细胞中 Bmi-1 的表达,以观察其对 K562 细胞生长的抑制作用及其对细胞周期、p16 蛋白表达的影响,从而探讨反义 Bmi-1 抑制白血病细胞增殖的作用机理,为白血病的治疗提供新的思路。 方法 1. 根据 Bmi-1 基因已知序列设计合成一对引物,上下游引物分别加入 XhoI 和 ClaI 的酶切位点,用反转录 PCR(RT-PCR)方法从人红白血病细胞株(K562)总 RNA 中扩增编码 Bmi-1 的基因片段,插入克隆载体 pMD18-T,构建 pMD18-Bmi-1 载体,经 XhoI,ClaI 双酶切出 Bmi-1 基因片段,并将该片段克隆至载体 pLNCX2 中构建pLNCX2-Bmi-1 真核表达载体。2. 以 pLNCX2-Bmi-1 为模板,选择Bmi-1 基因起始密码子及编码环指结构的核苷酸片段,反向连接于pLNCX2 质粒上得到反义 Bmi-1 表达质粒。3. 在体外转染中通过脂质体法将质粒导入 K562 细胞中,以 G418 进行筛选得到阳性克隆。4. 以更多还原

【Abstract】 Objectives Leukemia is a malignant tumor which threatens the life of patients seriously. The current way of treating leukemia is to eliminate the proliferative leukemic cells, however, only very few leukemic stem cells were in their proliferative state and therefore, resist therapy. In order to prevent the leukemia in its source, identifying the gene determining the self-renewal of leukemic stem cells and finding a way to inhibit its expression would be new approach to stop leukemic cell proliferation. Bmi-1 plays a central role in normal and leukemia hematopoietic stem cells self-renewal. The proliferative potential of leukemic stem and progenitor cells lacking Bmi-1 is compromised and leukemic stem cells would have a progressive failure. We constructed a plasmid , antisense Bmi-1 plasmid then transferred it into K562 cells, and to see if it can inhibit the proliferation of K562 cells and upgrade activity of p16. Methods 1.Using the total RNA extracted from a human erythroleukemia cell line (K562) as the template, the Bmi-1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers containing the restriction sites of XhoI and ClaI. The amplified fragment of Bmi-1 gene digested with XhoI and ClaI was subcloned into cloning vector pMD18-T and then into expression vector pLNCX2. 2.An antisense plasmid was constructed by reverse designing of PCR primers, and the PCR products were subsequently cloned to plasmid pLNCX2. 3.G418 was added into the medium after the plamid was successfully introduced into K562 cells by using lipofectactin- mediated DNA transfection. 4.The effects of on the proliferation of K562 cells were analyzed by means of trypanblue staining、MTT and colony forming. 5.Cell cycle was analyzed by cytometric. 6.The p16 activity of K562 cells was studied by immune chemistry methods. 7. Apoptosis was analyzed by DNA ladder. Results 1. The growth rate of antisense Bmi-1 transfected K562 cells was significantly slower than those of the controls. In contrast to control cells the colony forming ability of antisense Bmi-1 gene transfected cells decreased significantly (p<0.01). However, the survival rate of antisense K562 cells have no significantly difference when compared with empty plasmid K562 cells and parental K562 cells. 2. Cell cycle analysis using flow cytometry showed that percentage of antisense K562 cells in G0/G1 increased significantly when compared with empty plasmid K562 cells and parental K562 cells. 3. The p16 activity of antisense Bmi-1 transfected cells was significantly upgrade than those of controls. 4. The result of DNA ladder is negative and the experimental cells have no significantly apoptosis. Conclusion Antisense Bmi-1 can inhibit the growth of K562 cells and upgrade activity of p16 in vitro.更多还原