【作者】 李蕾；

【导师】 邹萍；

【作者基本信息】 华中科技大学 ， 血液内科， 2008， 博士

【摘要】 第一部分THP-1细胞中MLL-AF9融合基因的融合位点序列鉴定【目的】从急性单核白血病细胞系THP-1中克隆出MLL-AF9融合基因断裂点序列,并对其进行序列测定分析。【方法】利用RT-PCR方法从急性单核白血病细胞系THP-1中扩增出MLL-AF9融合基因断裂点附近序列,并用T-A克隆方法将其克隆至T载体上,经酶切鉴定后,进行序列测定分析。【结果】从急性单核白血病细胞系THP-1克隆出了MLL-AF9断裂点附近序列,大小为528bp,测序后示MLL基因断裂点位于外显子9,与AF9基因外显子5发生融合。【结论】本实验成功克隆了THP-1细胞系MLL-AF9断裂点的cDNA序列,为针对该序列设计特异性的siRNA进行基因沉默实验做准备。第二部分抑制MLL-AF9融合基因对THP-1细胞增殖的影响【目的】研究MLL-AF9融合基因对急性单核白血病细胞系THP-1细胞增殖的影响。【方法】选择THP-1细胞特有的MLL-AF9融合基因为靶基因,设计并合成siRNA片段。以人急性单核白血病细胞系THP-1为靶细胞,应用脂质体转染方法,将siRNA导入细胞,通过流式细胞仪检测siRNA转染效率,RT-PCR法比较转染前后MLL-AF9 mRNA表达水平的变化,MTT法检测细胞增生抑制率,并通过Hoechst33258染色观察siRNA作用于THP-1后细胞凋亡形态特征,流式细胞仪检测细胞凋亡水平和细胞周期的改变。【结果】siRNA转染效率为69.1%±1.8%,转染siRNA后MLL-AF9基因表达量大幅度减少(P<0.01),THP-1生长受到明显抑制,G0/G1期细胞增多(P<0.01),S期细胞减少(P<0.01),并使细胞周期阻滞于G0/G1期;凋亡小体增多,凋亡水平增加(P<0.01),而对照组则无上述表现(P>0.05)。【结论】MLL-AF9融合基因维持和促进人急性单核白血病细胞THP-1的增殖。第三部分MLL-AF9融合基因对THP-1细胞p27表达调控的影响【目的】研究MLL-AF9融合基因对人急性单核白血病细胞系THP-1的p27表达及转录调控的影响。【方法】选择THP-1细胞特有的MLL-AF9融合基因为靶基因,设计并合成siRNA片段。应用脂质体转染方法,将siRNA导入细胞,通过流式细胞仪检测siRNA转染效率,RT-PCR法比较转染前后MLL-AF9 mRNA表达水平的变化,Western Blot检测MLL-AF9蛋白水平沉默效果,比较细胞周期抑制蛋白p27表达变化,利用染色质免疫沉淀技术(Chromatin Immunoprecipitation ,ChIP)研究MLL-AF9融合蛋白是否与p27启动子结合。【结果】转染siRNA后THP-1细胞MLL-AF9表达明显受抑(P<0.01),p27表达上调(P<0.01),应用染色质免疫沉淀技术结合PCR扩增的方法分析发现MLL-AF9融合蛋白可与THP-1细胞p27基因启动子区域的DNA片段结合。【结论】急性单核白血病细胞系THP-1中的MLL-AF9融合蛋白通过与p27启动子的结合下调p27基因的表达。更多还原

【Abstract】 PART I Identification on the Sequence of MLL-AF9 Fusion Gene in Acute Monocytic Leukemia Cell Line THP-1Objective: Clone the MLL-AF9 fusion gene from the acute monocytic leukemia cell line THP-1 and analyze its junction sequence.Methods: RT-PCR was used to separate MLL-AF9 fusion gene from the acute monocytic leukemia cell line THP-1 and clone it into T vector for sequence analysis.Results: MLL-AF9 fusion gene has been cloned from the acute monocytic leukemia cell line THP-1. Sequencing showed that the fusion gene was a direct in-frame fusion between exon 9 of MLL and exon 5 of AF9.Conclusions: Clone and sequence the MLL-AF9 fusion gene from THP-1 successfully. It is necessary to design the siRNA targeted MLL-AF9 in the next part. PART II Effect of Down-regulating MLL-AF9 Fusion Gene on the Proliferation of Acute Monocytic Leukemia Cell Line THP-1Objective: To study the effect of MLL-AF9 fusion gene on the proliferation of human acute monocytic leukemia cell line THP-1.Methods: One group of siRNA was designed targeting MLL-AF9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Cell proliferation rate was analyzed by MTT assay. Hoechst33258 staining was used to observe the apoptotic bodies in the cells. The change of apoptosis rate and cell cycles was detected by flow cytometry.Results: SiRNA transfection efficiency was 69.1%±1.8%. The level of MLL-AF9 mRNA expression was significantly inhibited in siRNA-treated cells as compared with that in the controls (P<0.01). The percentage of G0/G1 phase cells was significantly increased in siRNA-treated cells in comparion with that in the control cells (P<0.01), but the percentage of S phase cells was significantly decreased (P<0.01). MLL-AF9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. Staining of the siRNA-treated cells with Hoechst 33258 showed the morphology characteristic to apoptosis.Conclusions: MLL-AF9 fusion gene sustains and promotes the proliferation of human acute monocytic leukemia cell line THP-1. PART III MLL-AF9 Fusion Gene Regulates Cyclin-Dependent Kinase Inhibitor p27 Expression in Acute Monocytic Leukemia Cell Line THP-1Objective: To explore whether MLL-AF9 oncegene regulates p27 expression in THP-1 cell line.Methods: SiRNA targeting MLL-AF9 were designed and constructed, and transfected into THP-1 by lipofectmine. Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was examined by reverse transcription polymerase chain reaction (RT-PCR) and the expression of MLL-AF9 and p27 protein was detected by Western blot. Chromatin immunoprecipitation (ChIP) assays was used to confirm whether MLL-AF9 binds to the p27 promoter in THP-1 cell line.Results: The level of p27 expression shows up-regulation at both the mRNA level and protein level after MLL-AF9 expression was significantly inhibited in siRNA-transfected cells as compared with that in the controls (P<0.01). MLL-AF9 binds to the p27 promoter in THP-1 cell line.Conclusion: MLL-AF9 oncoprotein in THP-1 cells inhibits the p27 expression, and regulates p27 promoter activity.更多还原