【作者】 陈磊；

【导师】 奚绪光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 正常的造血过程有赖于各种调控因素之间的平衡,正调控因素过多或负调控因素过少,均会造成造血失控。如果造血细胞或其子代细胞进行恶性性增生,即成为血液系统的恶性肿瘤。70年代中期,主要使用抑制恶性细胞增殖的化疗药物对白血病进行治疗,然而,白血病细胞除了恶性增殖外还具有例如分化受阻,凋亡失调和转移的生物学特征。在急性早幼粒细胞性白血病(acute promyelocytic leukemia,APL)病人骨髓中非正常早幼粒细胞积聚的特征明显的表明粒细胞分化受阻。自从1986年我国学者首先使用全反式维甲酸(all-trans retinoic acid,ATRA)分化治疗APL以来,ATRA已成为抗肿瘤药物并且功能上作为一种分化物在APL的治疗中表现出了很好的效果,ATRA的抗增殖活性主要来自于对增长抑制以及诱导分化,此外,在不同的细胞系中ATRA除具有抑制增殖作用外还能促进细胞的凋亡。维甲酸诱导基因-I(Retinoic acid-inducible gene-I, Rig-I)是含DExD/H box的RNA解旋酶,由925个氨基酸组成,N端包含2个CARD结构域(caspase recruitment domains),C端有1个RNA解旋酶结构域。Rig-I在先天性免疫中发挥重要作用,它可以识别细胞内的病毒RNA,并通过活化IRF3和NF-κB诱导I型干扰素(Type I interferons, IFNs)的产生,目前已成为免疫学领域的研究热点。然而Rig-I最早是在无病毒感染下ATRA诱导APL细胞株NB4分化时分离到的一个受调表达的基因,其与ATRA诱导的粒系分化通路密切相关。与其在抗病毒天然免疫方面的研究相比,到目前为止,Rig-I在造血细胞中的结构与功能以及信号传导方面的研究仍然不是很清楚。特别是与APL细胞系NB4(AML-M3型)同为急性髓系白血病(acute myeloid leukemia, AML)细胞系的人急性单核白血病细胞系U937(AML-M5型)在ATRA和干扰素(interferon, IFN)作用下Rig-I的功能及信号通路的研究未见报道。本实验首先在制备了小鼠Rig-I和人的Rig-I-N抗体并分别建立了诱导表达人Rig-I和调控Rig-I沉默的U937细胞基础上,研究ATRA和IFN对U937细胞增殖、分化、凋亡和周期影响并在此过程中对Rig-I的功能和信号通路进行了研究。主要得出以下结论:1.分别将小鼠Rig-I全长基因和人Rig-I-N 850bp序列构建到了原核表达载体,在大肠杆菌中得到了表达,纯化了小鼠Rig-I和人Rig-I-N的融合蛋白,并以此为抗原分别制备了特异性良好的多克隆抗体,制备的抗体能够识别在原核和真核表达系统中表达的各自抗原蛋白,并结合小鼠脾脏细胞Western blot分析和U937细胞免疫荧光技术表明制备的小鼠Rig-I和人Rig-I-N多克隆抗体具有良好的免疫特异性。2.通过PCR技术将人Rig-I基因构建到Tet-Off诱导表达系统的反应质粒pTRE2 -hyg中,转入293(Tet-Off)细胞验证Rig-I的表达后电转入U937(Tet-Off)细胞,筛选混合克隆细胞,对其单克隆化,Western blot挑选出撤Tetracycline后能够很好的诱导表达Rig-I的单克隆并结合细胞免疫荧光技术验证Rig-I的表达,得到了利用Tet-Off诱导表达系统表达Rig-I的U937细胞。3.将设计好的两条人Rig-I基因shRNA miR-30结构不完全序列PCR扩增为完全miR-30结构序列后构建到慢病毒表达载体p201中,在293T细胞中表明其具有很明显的RNAi效果,将其构建到Tet-Off系统中带有GFP的反应质粒pTRE-GFP-hyg后转入U937(Tet-Off)细胞,筛选出稳定混合克隆细胞并对其单克隆化,利用报告基因GFP表达及Western blot方法挑选RNAi作用明显的单克隆细胞并验证其RNAi的调控性,建立了基于Tet-Off系统调控的稳定Rig-I基因RNAi的U937细胞。4.ATRA、IFNα、IFNγ单独使用对U937细胞的增殖起到抑制作用,组合使用,特别是ATRA和IFNα合用显著的抑制其增殖,细胞周期分析也表现出相同的结果,绝大部分阻滞在G0/G1期;ATRA单独使用对细胞分化作用明显,ATRA和IFNα合用对分化和凋亡有协同作用。因此,ATRA和IFNα合用对于U937增殖抑制、分化、凋亡和周期阻滞具有协同增效的作用。5.U937单用ATRA和IFNγ对Rig-I诱导表达不明显,而单用IFNα对Rig-I诱导显著,尤其是ATRA和IFNα合用对Rig-I的诱导表达具有协同作用。与在ATRA和IFNα合用下诱导Rig-I基因上调表达的U937细胞相比,在相同条件下Rig-I沉默的U937细胞呈现相反的结果,表现为增殖明显、分化、凋亡减少,周期阻滞部分解除。正反两方面研究结果说明Rig-I的表达上调参与了ATRA和IFNα合用诱导U937细胞增殖抑制、分化、凋亡和周期阻滞。6.对细胞信号通路的研究表明ATRA和IFNα合用诱导Rig-I表达能够抑制AKT和AKT-mTOR通路下游P70和4EBP1的磷酸化活化,并抑制AKT下游另一靶蛋白FOXO3α转录因子磷酸化,促进P27周期阻滞蛋白的表达,而在相同条件下Rig-I沉默的U937细胞促进AKT及其通路的活化,而AKT及AKT信号通路对肿瘤细胞的增殖、分化、凋亡和周期起着决定性的作用,因此,Rig-I负调控AKT通路参与ATRA和IFNα诱导的U937细胞增殖抑制、分化、凋亡和周期阻滞。更多还原

【Abstract】 The normal regulation of hematopoiesis depends on the balance among various factors, regulatory factors are too many or negative regulatory factors are too few will cause out of control of hematopoiesis. If the hematopoietic cells or their progeny cells are malignant hyperplasia, it becomes the malignancy of blood system. Mid-70s, mainly using chemotherapeutic drugs of inhibiting the proliferation of malignant cells to treat the leukemia, In addition to malignant proliferation, leukemic cells have the biologic characteristics of blocked differentiation, apoptosis disorders and metastasis. In APL (acute promyelocytic leukemia) patients, unnatural accumulation of promyelocytic cells in bone marrow show the characteristics of granule cells significantly blocked. Since 1986, Chinese scholars firstly using ATRA (all-trans retinoic acid) to treat APL, ATRA has become an antitumor drug and as a material of differentiation therapy has demonstrated very good results in the treatment of APL. anti-proliferation activity of ATRA mainly comes from the growth inhibition and induction of differentiation, moreover, in addition to inhibit the proliferation, ATRA also promotes apoptosis in different cell lines.Rig-I (Retinoic acid-inducible gene-I) is a RNA helicase with DExD / H box, composed with the 925 amino acid residues, N terminal contains 2 CARD domain (caspase recruitment domains), C terminal has an RNA helicase domain. Rig-I plays an important role in innate immunity, it can identify the virus RNA within the cell, and induce the production of IFNs (typeⅠinterferon through the activation of IRF3 and NF-kB, now has become a hot topic in immunology. However Rig-I was firstly isolated from NB4 of APL cell line which is induced differentiation with ATRA in the absence of virus infection, and is closely related with the ATRA-induced granulocytic differentiation pathways. Compared with the research of anti-virus in innate immunity. So far, it is still not clear of the structure and function, signal transduction of Rig-I in hematopoietic cell. Especially, It has not report about the function and signaling pathway of Rig-I in U937 (human acute monocytic leukemia cell line; AML-M5) belonging to AML (acute myeloid leukemia) with NB4 (APL cell line; AML-M3) under the action of ATRA and IFN (interferon). In this study, first of all, on the basis of preparation of the mouse and human Rig-I antibody, establishing the induced expression of human Rig-I and regulatory Rig-I RNAi U937 cell lines separately, studied the effect of cell proliferation, differentiation, apoptosis and cell cycle on U937 under the action of ATRA and IFN, in the process of above, Rig-I function and signaling pathways have been studied.The main results were as follows:1. The mRig-I (full-length gene of Rig-I in mouse) and hRig-I-N (850 bp sequence of human Rig-I N terminal) were built into the prokaryotic expression vector, expressed in E. coli, fusion proteins of mRig-I and hRig-I-N were purified and separately prepared for the polyclonal antibodies of good specificity, the prepared antibodies can identify the expression of respective antigens from prokaryotic and eukaryotic expression system and Combined with mouse spleen cells and immunofluorescence technique of U937 cells indicate that polyclonal antibodies of mRig-I and hRig-I-N have a good immune specificity.2. By PCR, hRig-I gene was constructed into the pTRE2-hyg response plasmid of the Tet-Off inducible expression system, after transformed into 293 (Tet-Off) cells and verified the expression of Rig-I, the constructed plasmid was electroporated into the U937 (Tet-Off) cells, cells were selected and isolated, after withdrawal of Tetracycline, clones which can be well induced expression of Rig-I were picked by Western and combined with immunofluorescence verified Rig-I expression, the U937 can be induced expression of Rig-I was obtained by using Tet-Off inducible expression system.3. Two designed shRNA miR-30 partial sequences of human Rig-I were amplify to complete sequences of miR-30 structure and constructed into the p201 lentiviral vector, after indicated have obvious RNAi effect in 293T, the two complete shRNA sequences were built into response plasmid of pTRE2-GFP-hyg and electroporated into the U937 (Tet-Off) cells, these cells were selected and isolated, clones were picked and verified the RNAi regulation by using the expression of GFP report gene and Western blot technique, the regulatable Rig-I RNAi stable U937 was established based on the Tet-Off system.4. ATRA, IFNα, IFNγalone inhibited the U937 cell proliferation, in combination, in particular, ATRA and IFNαcombination significantly inhibited the cell proliferation, cell cycle analysis also showed the same result, most blocked in the G0/G1 period; ATRA alone has obvious effect on cell differentiation, ATRA and IFNαcombination have the synergy effect on differentiation and apoptosis. Therefore, ATRA and IFNαcombination show a synergistic effect on the proliferation inhibition, differentiation, apoptosis and cell cycle arrest of U937. 5. The Rig-I expression of U937 induced by ATRA or IFNγalone is not obvious, but the induction of the Rig-I is significantly by IFNαalone, especially it was show a synergistic effect on the induced expression of Rig-I by ATRA and IFNαcombination. Compared with the upregulated expression of Rig-I in U937 combination ATRA with IFNα, under the same conditions, Rig-I silent U937 cells showed the opposite results, showing significant proliferation, differentiation and apoptosis reduction, cycle arrest partially relieved. Both positive and negative Research showed that upregulated expression of Rig-I involved in proliferation inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells induced by ATRA and IFNαcombination.6. Research on the cell signaling pathway of U937 showed that Rig-I expression induced by the combination of ATRA and IFNαcan inhibit the phosphorylation and activation of not only AKT but also P70 and 4EBP1 which is the downstream of AKT-mTOR pathway, also inhibit the phosphorylation of transcription factor FOXO3α, another AKT downstream target protein, to promote P27 cycle arrest protein expression, Under the same conditions the silence of Rig-I can promote activation of AKT and its downstream pathway, The AKT and AKT signaling pathway play a decisive role on proliferation, differentiation, apoptosis and cell cycle of tumor cell, therefore, through negative regulation of AKT pathway Rig-I involved in ATRA and IFNαinduced cell proliferation inhibition, differentiation, apoptosis cell cycle arrest of U937.更多还原