【作者】 刘辉；

【导师】 金洁； 钱文斌；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 白血病是一种克隆性起源,多能干细胞或早期的祖细胞(髓系或淋系)突变而引起的造血系统恶性肿瘤。白血病细胞恶性增殖,进入血循环、并浸润全身各组织脏器;临床可见不同程度的贫血、出血、感染发热以及肝、脾、淋巴结肿大和骨骼疼痛等浸润症状。目前白血病已经成为我国最常见恶性肿瘤之一,发病率在各种肿瘤中占第六位,而且在年轻人恶性疾病中排名首位。尽管通过传统细胞毒药物联合化疗、维甲酸和砷剂诱导分化治疗、造血干细胞移植和支持治疗等综合方法,已经使急性白血病的完全缓解率达到70-90%,3年无病生存率达40-50%。但仍存在化疗相关毒性、化疗药物的非选择性以及对复发难治白血病的耐药性等诸多问题,促使研究者致力于研究各种新型的、更具靶向性的治疗手段,而基因病毒治疗正是其中新兴的一个研究方向。溶癌腺病毒在肿瘤细胞中复制后可以溶解细胞,并释放出子代病毒颗粒,进一步感染周边的肿瘤细胞,直至完全消灭肿瘤。本研究中,我们构建新型溶癌腺病毒,具有5型和35型嵌合纤毛或5型和11型嵌合纤毛,并且E1B-55kDa缺失,同时可以携带TRAIL或自噬相关基因BECN等效应基因,从而达到基因治疗与病毒治疗相结合的策略。此外,我们进一步设计和研究化疗、基因治疗和病毒治疗三种手段相结合杀伤白血病细胞这一新思路,以期为临床白血病治疗提供新的方法和策略。本研究分三部分进行:第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究;第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究;第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究。第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究目的:构建新型溶癌腺病毒SG235,其具有5型和35型嵌合纤毛并且E1B-55kDa缺失,同时可以携带TRAIL表达框;通过体内外实验证实和比较SG235及SG235-TRAIL抗白血病细胞效应,并探讨其作用机制。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。方法:构建新型溶癌腺病毒SG235、SG235-增强绿色荧光蛋白(enhanced greenfluorescence protein,EGFP)以及SG235-TRAIL,通过荧光倒置显微镜、流式细胞术、病毒滴度检测和western blot法检测E1A的手段,来研究SG235对白血病细胞株、淋巴瘤细胞株、多发性骨髓瘤(multiple myeloma,MM)细胞株、患者原代白血病细胞和白血病细胞集落(leukemic colony forming unit,CFU-L)的感染能力及其复制活性;通过western blot法来明确SG235 E1B-55kDa是否缺失;通过四甲基偶氮唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphemyltetra-zolium bromide,MTT)法检测各型腺病毒对白血病细胞株的细胞毒作用;通过末端脱氧核苷酸转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记(terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick end labeling,TUNEL)法和磷脂酰丝氨酸(phosphatidylserine,PS)转位法来证实SG235、SG235-TRAIL诱导白血病细胞株和患者原代白血病细胞发生凋亡;通过罗丹明123(rhodamine 123,Rh123)染色法来检测SG235-TRAIL感染白血病细胞株后线粒体膜电位改变;采用western blot法来检测各型腺病毒感染白血病细胞株和患者原代白血病细胞后Caspase家族蛋白、B细胞白血病/淋巴瘤因子-2(B cell leukemia/lymphoma,Bcl-2)家族蛋白的改变,并进一步加用Caspase-3抑制剂(z-DEVD-FMK)和Caspase-8抑制剂(z-IETD-FMK)来观察其对SG235-TRAIL感染白血病细胞株后诱导凋亡水平的影响;以各型腺病毒作用Hela细胞,通过结晶紫染色法和western blot法检测Caspase家族蛋白来明确SG235和SG235-TRAIL是否具有杀伤实体瘤细胞株的能力;收集临床确诊白血病患者骨髓液,检测原代白血病细胞的CD46表达情况;通过CFU-L和粒细胞-巨噬细胞集落(giranulocyte-macrophage colony forming unit,GM-CFU)形成能力实验来证实各型腺病毒是否具有选择性杀伤白血病细胞的特性;通过酶标记免疫吸附测定(enzyme-labeled immunosorbent assay,ELISA)法来检测各型腺病毒感染细胞后上清液TRAIL水平;通过观察各型腺病毒对Kasumi-1细胞荷瘤小鼠瘤体生长的影响以及TUNEL染色法来研究各型腺病毒在体内杀伤白血病细胞的作用。结果:(1)收集不同类型白血病患者原代白血病细胞122例,CD46总体阳性率为78.7%。表明以CD46作为受体的腺病毒载体SG235具备了感染白血病细胞的前提条件;(2)SG235体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈时间依赖性和剂量依赖性;(3)SG235本身能够诱导白血病细胞株发生凋亡,激活Caspase-9、Caspase-3和多聚腺苷二磷酸核糖聚合酶(poly-(adenosinediphosphate -ribose)polymerase,PARP),并具有剂量依赖性;(4)SG235-TRAIL和SG235都对白血病细胞株具有明显细胞毒作用,并具有剂量依赖性,而SG235-TRAIL较SG235细胞毒作用更显著;(5)SG235-TRAIL能够诱导白血病细胞株发生凋亡,且诱导凋亡效应较SG235更强;(6)SG235-TRAIL感染白血病细胞株中Caspase-8及其作用底物Bcl-2同源结构域3结构域凋亡诱导蛋白(Bcl-2homology 3 interacting domain death agonist,BID)激活,Bcl-2相关X蛋白(Bcl-2associated X protein,BAX)上调,线粒体膜电位下降,而Caspase-9和Caspase-3也可见激活;(7)SG235-TRAIL诱导白血病细胞株凋亡的效应可以被Caspase-3抑制剂与Caspase-8抑制剂部分抑制,其中Caspase-3抑制剂作用更为明显;(8)SG235与SG235-TRAIL感染后,Kasumi-1细胞Bcl-2与粒细胞白血病序列-1(myeloid cell leukemia sequence-1,MCL-1)表达下调,而Bcl-2相互作用细胞死亡介导因子(Bcl-2 interacting mediator of cell death,BIM)、Bcl-2同源拮抗物(Bcl-2homologous antagonist/killer,BAK)和Bcl-2样蛋白(Bcl-2 like protein,Bcl-XL)的表达未见明显改变;(9)SG235与SG235-TRAIL对实体瘤细胞株也有杀伤效应;(10)SG235以及携带TRAIL表达框的SG235-TRAIL能够有效感染原代白血病细胞,诱导其发生凋亡,激活Caspase-8、Caspase-3及PARP,并有效杀伤原代白血病细胞,而SG235-TRAIL作用较SG235更显著;(11)SG235与SG235-TRAIL能够有效感染CFU-L,并选择性抻制CFU-L形成,而SG235-TRAIL几乎完全抑制CFU-L的形成,此外,SG235与SG235-TRAIL不抑制或轻度抑制正常GM-CFU形成;(12)SG235-TRAIL感染白血病细胞株后表达TRAIL的水平要明显高于作为载体对照的Ad5/35-TRAIL组,SG235未见明显TRAIL表达。此外,SG235-TRAIL表达TRAIL水平呈时间依赖性;(13)SG235能显著抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而ZD55几乎不影响瘤体生长;(14)SG235-TRAIL较SG235更能显著地抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而且小鼠瘤体内凋亡细胞比例更高,而作为载体对照的Ad5/35-TRAIL没有表现出抗肿瘤效应。第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究目的:根据化疗、基因治疗和病毒治疗三种手段相结合杀伤白血病细胞这一新思路,以SG235-TRAIL与HHT联合作用白血病细胞,并通过体外实验证实细胞毒作用并分析两者的协同效应,并进一步探讨其作用机制以及对正常细胞的杀伤效应,以期为临床白血病治疗提供新的方法和策略。方法:通过MTT法和乳酸脱氢酶(lactate dehydrogenase,LDH)法检测SG235-TRAIL与HHT对白血病细胞株的细胞毒作用,并通过Chou-Talalay联合指数法进行分析;通过PS转位法检测SG235-TRAIL与HHT诱导白血病细胞株凋亡;采用western blot法来检测SG235-TRAIL与HHT作用白血病细胞株后E1A、TRAIL、Caspase家族蛋白和Bcl-2家族蛋白的改变;通过MTT法检测SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应;体外培养和鉴定间充质干细胞(mesenchymal stem cells,MSCs),并通过结晶紫染色法检测HHT和SG235-TRAIL对MSCs的细胞毒作用。结果:(1)SG235-TRAIL与HHT在体外联用杀伤白血病细胞株,表现出不同程度的协同效应;(2)SG235-TRAIL与HHT能协同诱导白血病细胞株凋亡;(3)SG235-TRAIL对白血病细胞株的感染能力及其复制水平没有因为HHT的联用而发生改变;(4)SG235-TRAIL与HHT联用作用Kasumi-1细胞,其TRAIL蛋白表达水平高于SG235-TRAIL单用;(5)SG235-TRAIL以激活Caspase-8为主,HHT以激活Caspase-9为主。而SG235-TRAIL与HHT联用能较大程度激活Caspase-8及其作用底物BID、Caspase-9、Caspase-3和PARP,同时下调Bcl-2和Mcl-1的表达;(6)SG235-TRAIL与HHT联用,白血病细胞株Bcl-2与Mcl-1表达下调;(7)SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应较小,具有靶向性。第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究目的:构建新型溶癌腺病毒SG511-BECN,其具有5型和11型嵌合纤毛并且E1B-55kDa缺失,同时携带BECN表达框,通过体内外实验证实SG511-BECN通过诱导白血病细胞自噬性死亡而发挥抗白血病细胞效应。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。方法:构建新型溶癌腺病毒SG511、SG511-EGFP以及及SG511-BECN,通过荧光倒置显微镜、流式细胞术和western blot法检测beclin-1的手段,来研究SG511对白血病细胞株、淋巴瘤细胞株、MM细胞株、原代白血病细胞和CFU-L的感染能力及其复制活性;通过结晶紫染色法检测SG511-BECN对实体瘤细胞株的细胞毒作用,通过MTT法检测SG511-BECN对恶性血液病细胞株的细胞毒作用;通过吖啶橙染色法、透射电镜和微管相关蛋白1轻链3(light chain 3,LC3)-GFP-K562细胞荧光改变检测SG511-BECN诱导白血病细胞株自噬,并进一步通过western blot法检测SG511-BECN感染K562细胞后LC3和p62表达改变;通过观察各型腺病毒对K562细胞荷瘤小鼠瘤体生长的影响来研究各型腺病毒在体内杀伤白血病细胞的作用。结果:(1)SG511体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈剂量依赖性和细胞特异性;(2)SG511体外实验中能够有效感染CFU-L;(3)SG511-BECN能有效感染K562细胞,并表达beclin-1蛋白;(4)SG511-BECN和SG511在体外都对实体瘤细胞株、恶性血液病细胞株具有明显细胞毒作用,而SG511-BECN较SG511细胞毒作用更显著;(5)SG511-BECN在体外能够诱导白血病细胞株发生自噬,并伴随LC3-Ⅱ的表达和p62的降解;(6)SG511-BECN和SG511显著地抑制K562细胞荷瘤小鼠瘤体生长,而SG511-BECN较SG511细胞毒作用更显著。结论:SG235和SG511作为两种新型的具有嵌合纤毛的溶癌腺病毒,能有效感染白血病细胞株和原代白血病细胞,而携带了效应基因的SG235-TRAIL,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生凋亡;同样,携带了效应基因的SG511-BECN,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生自噬性死亡。此外,SG235-TRAIL联合HHT能够发挥协同抗白血病效应。更多还原

【Abstract】 Leukemia is a series of clonal hematopoietic malignancies caused by mutations of stem cells or early progenitor cells(myeloid or lymphoid).Malignant proliferation of leukemic cells makes themselves into the circulation,infiltrating organs and tissues. Varying degrees of anemia,bleeding,fever can be observed as well as hepatomegaly, splenomegaly,bone pain and so on.So far,leukemia has become one of the most common malignancies in China with the incidence accounting sixth place,and leukemia has become the most common malignant disease among young people. Despite the integrated adoption of traditional combined chemotherapy,retinoic acid and arsenic trioxide-induced therapy,hematopoietic stem cell transplantation and supporting therapy has resulted in a complete remission rate of 70-90%and a 3-year disease-free survival rate of 40-50%,there are still chemotherapy-related toxicity, non-selectivity as well as drug resistance of relapsed or refractory leukemia.These questions prompt researchers to study for a variety of novel target therapies,while the gene-viral therapy is a important one among them.Oncolytic adenoviruses replicate in tumor cells and cause cell lysis,and then they release themselves to further infect surrounding tumor cells,until the total eradication of the tumor.In this study,we constructed novel oncolytic adenoviruses of ElB-55kDa deletion with chimeric Ad5/35 fibers or chimeric Ad5/ll fibers.These adenoviruses.can also carry TRAIL gene or BECN gene,so as to achieve the combination of gene therapy and viral therapy.In addition,we further designed chemo-gene-viral therapy in order to provide novel strategy for leukemia clinical practice.This study is divided into three parts:PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells;PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells;PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on leukemic cells.PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cellsObjective:We constructed novel oncolytic adenovirus SG235 of E1B-55kDa deletion with a chimeric Ad5/35 fibers,and the adenovirus can also carry TRAIL cassette.We further confirm SG235 and SG235-TRAIL anti-leukemic cell effect by in vitro and in vivo experiments,and explore the mechanism.By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.Methods:We first constructed the adenovirus SG235,SG235-EGFP and SG235-TRAIL,and study the infection and replication ability of SG235 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry,viral titer testing and western blot for E1A;confirm the ElB-55kDa deletion of SG235 via western blot; confirm cytotoxicity of different adenoviruses on leukemia cell lines via MTT;prove SG235 and SG235-TRAIL inducing apoptosis on leukemia cell lines and primary leukemia cells via TUNEL and PS translocation;detect the mitochondrial membrane potential change of leukemia cell lines after SG235-TRAIL infection via Rh123 staining;use western blot to detect Caspase family proteins,Bcl-2 family proteins after various types of adenoviruses infection in leukemia cell lines and primary leukemia cells;add Caspase-3 inhibitor(z-DEVD-FMK) and Caspase-8 inhibitor (Z-IETD-FMK) to observe the apoptosis levels induced by SG235-TRAIL;observe SG235 and SG235-TRAIL anti-tumor effect on Hela cell line via crystal violet staining and western blot for Caspase family;collect bone marrow cells from clinically diagnosed leukemia patients to detect the expression of CD46;to determine the selective cytotoxicity of adenoviruses on leukemia cells via CFU-L and GM-CFU forming test;detect the supernatant TRAIL levels after adenoviruses infection via ELISA;observe the various types of adenoviruses on tumor growth of Kasumi-1 xenograft-bearing SCID mice and observe the in vivo anti-leukemic effect of adenoviruses by TUNEL staining.Results:(1) 122 cases of leukemia cells is collected,the overall CD46-positive rate is 78.7%,indicating that SG235 can effectively infect leukemia cells;(2) SG235 can effectively infect leukemia,lymphoma and MM cell lines in vitro with time-dependent and dose-dependent manners;(3) SG235 can induce apoptosis in leukemia cell lines and activate Caspase-9,Caspase-3 as well as PARP with a dose-dependent manner;(4) both SG235-TRAIL and SG235 have obvious cytotoxicity on leukemia cell lines with a dose-dependent manner,while SG235-TRAIL is superior to SG235;(5) SG235-TRAIL can induce apoptosis in leukemia cell lines and is superior to SG235;(6) the leukemia cell line infected by SG235-TRAIL has Caspase-8 and its substrate BID activated,BAX expression increased,mitochondrial membrane potential lost as well as Caspase-9 and Caspase-3 activated;(7) SG235-TRAIL-induced apoptosis in leukemia cell lines can be inhibited by Caspase-3 and Caspase-8 inhibitors,while Caspase-3 inhibitor is superior to Caspase-8 inhibitor;(8) Bcl-2 and Mcl-1 is down regulated while BIM,BAK and Bcl-XL has no obvious change in Kasumi-1 cell lines after SG235 or SG235-TRAIL infection;(9) SG235 and SG235-TRAIL retain their cytotoxic effect on tumor cell lines;(10) SG235 and SG235-TRAIL can effectively infect primary leukemia cells, induce apoptosis,activate Caspase-8,Caspase-3 and PARP as well as kill primary leukemia cells,while SG235-TRAIL is superior to SG235;(11) SG235 and SG235-TRAIL can efficiently infect CFU-L,and selectively inhibit the formation of CFU-L,while SG235-TRAIL almost completely inhibits the formation of CFU-L,in addition,SG235 and SG235-TRAIL can not or slightly inhibit normal formation of GM-CFU;(12) the level of TRAIL expression is significantly higher after SG235-TRAIL infection than Ad5/35-TRAIL,while SG235 do not express TRAIL obviously;(13) SG235 can significantly inhibit the tumor growth in Kasumi-1 xenograft-bearing SCID mice while ZD55 can’t;(14) SG235-TRAIL is superior to SG235 in inhibiting tumor growth in Kasumi-1 xenograft-bearing SCID mice and inducing apoptosis in vivo,while Ad5/35-TRAIL shows no anti-tumor effect.PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cellsObjective:According to the thinking of chemo-gene-viral therapy,we combine SG235-TRAIL and HHT on leukemia cells,and confirm the cytotoxicity in vitro as well as conduct synergy analysis,furthermore,we explore the mechanism,with a view to provide new methods and strategies for clinical practice.Methods:We confirm the cytotoxic effect of combination of SG235-TRAIL and HHT via MTT and LDH test,then analyze the combination effect via Chou-Talalay method;SG235-TRAIL and HHT-induced apoptosis in leukemia cell lines is confirmed by PS translocation;E1A,TRAIL,Caspase family proteins and Bcl-2 family proteins is examined via western blot;SG235-TRAIL and HHT combination on normal bone marrow cells is tested via MTT;MSCs is cultured and identified,then HHT and SG235-TRAIL combination on MSCs is tested via crystal violet staining.Results:(1) SG235-TRAIL and HHT show anti-leukemia cell lines effect in vitro with different levels of synergy;(2) SG235-TRAIL and HHT synergistically induce apoptosis in leukemia cell lines;(3) the infection and and replication ability of SG235-TRAIL are not affected by HHT;(4) TRAIL expression level of SG235-TRAIL and HHT combination is more signigicant than SG235-TRAIL alone; (5) SG235-TRAIL mainly activates Caspase-8 and HHT mainly activates Caspase-9, Caspase-8 and its substrate BID,Caspase-9,Caspase-3 and PARP are significantly activated by SG235-TRAIL and HHT combination in leukemia cell lines;(6) Bcl-2 and Mcl-1 are down regulated by SG235-TRAIL and HHT combination in leukemia cell lines;(7) SG235-TRAIL and HHT combination has little effect on normal bone marrow cells.PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on Ieukemic cellsObjective:We constructed novel oncolytic adenovirus SG511 of ElB-55kDa deletion with chimeric Ad5/11 fibers,and the adenovirus can also carry BECN cassette.We further confirm SG511-BECN anti-leukemic cell effect by inducing autophagic cell death via in vitro and in vivo experiments,and explore the mechanism. By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.Methods:We first constructed the adenovirus SG511,SG511-EGFP and SG511-BECN,and study the infection and replication ability of SG511 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry and western blot for beclin-1;observe SG511 -BECN anti-tumor effect on tumor cell lines via crystal violet staining,and SG511-BECN anti-tumor effect on hematological malignant cell lines via MTT;detect autophagy induced by SG511-BECN via acridine orange staining, transmission electron microscopy and LC3-GFP-K562 cell fluorescence change; further observe the expression of LC3 and p62 via western blot in SG511-BECN-infected K562 cell line;in vivo study of SG511-BECN on the tumor growth of K562 xenograft-bearing SCID mice.Results:(1) SG511 can effectively infect leukemia,lymphoma and MM cell lines in vitro with a dose-dependent manner;(2) SG511 can efficiently infect CFU-L; (3) SG511-BECN can effectively infect K562 cell line,and express beclin-1;(4) SG511-BECN is superior to SG511 in killing tumor cell lines and malignant hematological cell lines;(5) SG511-BECN can efficiently induce autophagy in leukemia cell lines with LC3-Ⅱexpression and p62 degradation;(6) SG511-BECN is superior to SG511 in inhibiting tumor growth in K562 xenograft-bearing SCID mice.Conclusion:SG235 and SG511,as two viruses with novel chimeric fiber,can efficiently infect leukemia cell lines and primary leukemia cells.With TRAIL cassette, SG235-TRAIL can cause tumor cell lysis as well as induce apoptosis;with beclin-1 cassette,SG511-BECN can cause tumor cell lysis as well as induce autophagic cell death.In addition,SG235-TRAIL and HHT can exert their anti-leukemia effect synergistically.更多还原