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儿童急性白血病IGFBP-rP1基因的表达及机制探讨
The Mechanism of IGFBP-rP1 Gene in Childhood Acute Leukemias
【作者】 满孝蕊；
【导师】 胡绍燕；
【作者基本信息】 苏州大学 ， 儿科学， 2010， 硕士
【摘要】 一、儿童急性白血病骨髓中IGFBP-rP1基因表达及临床意义目的:定量检测IGFBP-rP1基因在儿童急性白血病骨髓细胞的表达水平,并与临床预后因素进行相关性分析,探讨其在儿童急性白血病发生的作用及临床意义。方法:利用实时定量RT-PCR(QRT-PCR)技术检测168例/次儿童急性白血病骨髓细胞中IGFBP-rP1基因的表达水平,并与同期30例非白血病患儿作对照。根据儿童急性白血病的预后因素,分析IGFBP-rP1基因与临床预后的相关性。结果:初诊组急性白血病患儿IGFBP-rP1基因表达水平明显高于非白血病患儿(M估计值分别为0.091、0.137);且急性髓细胞白血病(AML)初诊组高于急性淋巴细胞白血病(ALL)初诊组(P=0.0130),缓解时AML组表达最低,接近非白血病患儿组;复发时其表达水平再度升高(M估计值=0.084)。就ALL组而言,基因的表达水平与病程阶段无明显相关性(P值均>0.05);且IGFBP-rP1基因的表达水平与危险度评估无明显相关性(P>0.05)。结论: IGFBP-rP1可能参与小儿AML的发生、发展。二、IGFBP-rP1基因作用机制研究。目的:利用RNA干扰(RNAi)技术下调白血病细胞株U937细胞中IGFBP-rP1基因的表达,通过观察IGFBP-rP1基因对白血病细胞株U937细胞增殖、粘附、穿膜及侵袭等生物学行为的影响,初步探讨IGFBP-rP1基因在AML中的作用机制。方法:选择对数生长期的白血病细胞株U937,采用逆转录-聚合酶链式反应(RT-PCR)检测U937中IGFBP-rP1基因的表达;从上海吉玛制药技术有限公司购买三对IGFBP-rP1基因小干扰RNA(small interfering RNA, siRNA)。优化转染条件,筛选最有效的干扰序列,瞬时转染U937细胞。QRT-PCR方法和Western Blot方法验证干扰效果,并对IGFBP-rP1下调后的U937细胞进行细胞增殖、粘附、穿膜、浸润实验。结果: IGFBP-rP1下调后的U937细胞,24小时后细胞增殖能力受抑(0.580±0.159),明显低于未干扰组和阴性对照组(1.049±0.274;0.946±0.195)(P<0.01);转染靶向IGFBP-rP1基因siRNA的U937细胞其粘附能力明显低于未干扰组和阴性对照组(0.247±0.031 VS 0.406±0.023,0.395±0.011)(P<0.01);细胞穿膜及侵袭实验表明干扰IGFBP-rP1基因后的白血病细胞株U937穿膜及侵袭能力均较对照组减弱,[(0.387±0.021)×105VS(1.017±0.031)×105, (0.908±0.027)×105; (0.197±0.098)×105 VS (0.493±0.067)×105, (0.469±0.083)×105](P均<0.01),差异有显著性。结论:IGFBP-rP1基因可能通过改变细胞的增殖、粘附、穿膜、侵袭能力参与白血病发生发展过程。
【Abstract】 1 The significance of IGFBP-rP1 expression in childhood acute leukemiasObjective: To explore the role of insulin-like growth factor-binding protein related protein 1(IGFBP-rP1) gene in leukemogenesis and its significance in children acute leukemias.Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) method was used to detect the level of IGFBP-rP1 expression in bone marrow (BM) specimen from 168 children with acute leukemia (AL) at different stages and 30 non-leukemia patients as control. Meanwhile the relationship between the level of IGFBP-rP1 expression and clinical outcome was analyzed according to established clinical prognostic factors.Results: Expression level of IGFBP-rP1 at the initial diagnosed marrow specimen from acute leukemia children was significantly higher than that of non-leukemia children. (M-Estimators are 0.091,0.137 respectively])Expression level of IGFBP-rP1 in newly diagnosed acute myeloid leukemia (AML) is higher than that in newly diagnosed acute lymphocytic leukemia (ALL) (P =0.013), then reduced to less than the control group after complete remission and increased again at relapse(M-Estimator is 0.084). However, IGFBP-rP1 expression level didn’t show significant change among three stages in ALL treatment. And the expression level of IGFBP-rP1 has no significant correlation with the risk assessment(P>0.05).Conclusion: IGFBP-rP1 expression demonstrated a close relation with AML at presentation and after treatment which suggested that IGFBP-rP1 may play an important role in leukemogenesis of AML and could be a new MRD marker for AML treatment.2 The mechanism of IGFBP-rP1 gene in Leukemia CellsObjective: To explore the effect of IGFBP-rP1 gene on proliferation,adhesion,migration and invasion in U937 cells after the expression of IGFBP-rP1 was lowered using RNAi. The mechanism of IGFBP-rP1 was studied in AML.Methods: The expression of IGFBP-rP1 in U937 was confirmed by RT-PCR. Three pairs of double-strand siRNA targeting IGFBP-rP1 gene used in the following experiments were purchased from Shanghai GenePharma Co.,Ltd. Optimization in concentration and ratio was performed in order to screen the most effective sequence of siRNAs of IGFBP-rP1 for further experiment. QRT-PCR and Western Blot were used to detect the expression of IGFBP-rP1 in U937 cells after transiently transfected with siRNA of IGFBP-rP1. Cell proliferation, adhesion, transendothelial migration and invasion were performed in transfected cells and controls.Results: After transfected with siRNA of IGFBP-rP1 in U937 cells, the ability of cell proliferation was significantly decreased at 24h (0.580±0.159)compared to normal control and negative contro(l1.049±0.274、0.946±0.195 respectively)(P<0.01); transfected with siRNA targeting IGFBP-rP1 gene, the ability of cell adhesion was significantly lower than their control groups(0.247±0.031 vs 0.406±0.023,0.395±0.011)(P<0.01); cell membrane invasion assay showed that interfere with IGFBP-rP1 gene in U937 to wear invasive ability of membrane less than those in the control groups[(0.387±0.021)×105VS(1.017±0.031)×105, (0.908±0.027)×105; (0.197±0.098)×105 VS (0.493±0.067)×105, (0.469±0.083)×105](P<0.01), the difference was significant.Conclusion: IGFBP-rP1 gene may take part in the development and progression of leukemia by altering the cell proliferation, adhesion, transmembrane and invasion ability.