血管内皮细胞生长因子受体2基因短发夹RNA介导基因沉默对白血病的抑制(英文)

Inhibition of leukemia mediated by vascular endothelial growth factor receptor 2 short hairpin RNA gene silencing

背景:血管内皮细胞生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)在血管内皮细胞生长因子的信号转导中有主导效应,并在某些疾病包括白血病的病理性血管生成中起关键作用。另外,白血病细胞分泌的血管内皮细胞生长因子还诱导自身表达VEGFR2,从而维持自身存活、增殖。目的:观察表达VEGFR2短发夹RNA(short hairpin RNA,shRNA)的慢病毒载体Lenti6/shVEGFR2在全身性NOD/SCID白血病模型鼠中的作用。设计:随机、平行对照、开放性实验。单位:中山大学附属第二医院儿科,中山大学生物工程研究中心。材料:实验于2004-05/2006-01在中山大学附属第二医院医学研究中心、中山大学生物工程研究中心完成。分别以HL60,EA·hy926细胞株作为实验用白血病细胞、人血管内皮细胞的来源。Block-iT Lentiviral RNAi Expression System(Invitrogen);human VEGFR2 Mcb(PE标记,R&D)。CD31组化试剂盒(武汉博士德公司),CD33-PE流式细胞荧光标记抗体(BD公司)。方法:①制备并筛选VEGFR2基因的最有效siRNA,并据此设计shRNA寡核苷酸链。②构建pU6/shVEGFR2入门克隆。瞬时转染细胞,构建表达克隆Lenti6/shVEGFR2,与ViraPowerTM Packaging Mix共转染到293FTTM细胞中,产生携带表达克隆的慢病毒载体。③pU6/shVEGFR2入门克隆转染和Lenti6/shVEGFR2转导HL60细胞,MTT法测定HL60细胞抑制率。④建立HL60白血病模型后观察注入人血管内皮细胞后小鼠骨髓微血管的分布。⑤将NOD/SCID小鼠20只随机分为4组,每组5只:白血病模型鼠组,白血病模型鼠静脉注射重组慢病毒组,白血病模型鼠静脉注射重组慢病毒和内皮细胞组及白血病模型鼠静脉注射内皮细胞组。检测各组小鼠的骨髓细胞CD33表达变化、骨髓微血管密度变化及外周血细胞涂片、肝、脾的肿瘤浸润情况,以了解Lenti6/shVEGFR2重组慢病毒对小鼠白血病的作用。主要观察指标:①VEGFR2siRNA鉴定结果。②pU6/shVEGFR2入门克隆转染HL60后对其VEGFR2表达的影响。③pU6/shVEGFR2入门克隆转染、Lenti6/shVEGFR2表达克隆转导HL60细胞后细胞抑制率比较。④慢病毒介导的shRNA对HL60模型鼠VEGF/VEGFR2旁分泌和自分泌的影响。结果:①pU6/shVEGFR2入门克隆转染HL60后对其VEGFR2表达的影响:VEGFR2siRNAc抑制HL60细胞效率最高,利用其构建的pU6/shVEGFR2入门克隆并转染HL60,抑制细胞效率达84.9%;显著抑制HL60细胞VEGFR2基因mRNA和蛋白的表达。②pU6/shVEGFR2入门克隆转染、Lenti6/shVEGFR2表达克隆转导HL60细胞后细胞抑制率比较:pU6/shVEGFR2入门克隆转染、Lenti6/shVEGFR2表达克隆转导HL60细胞后48h细胞抑制率相近,48h后入门克隆转染组细胞抑制率明显下降(P<0.01);而表达克隆转导组细胞抑制率变化缓慢,在96h达高峰,120h稍降,变化无显著差异。③慢病毒介导的shRNA对HL60模型鼠VEGF/VEGFR2旁分泌和自分泌的影响:白血病模型组、白血病模型鼠静脉注射重组慢病毒组、白血病模型鼠静脉注射重组慢病毒和内皮细胞组小鼠骨髓流式细胞仪HL60检测结果分别为(25.8%±4.9)%,(14.3%±5.1)%,(8.4±2.6)%,三组比较,差异有显著性意义(P<0.05);白血病模型鼠静脉注射重组慢病毒和内皮细胞组微血管密度明显小于白血病模型鼠静脉注射内皮细胞组。白血病模型鼠静脉注射重组慢病毒和内皮细胞组与其他组相比HL60细胞数最低。结论:①pLenti6/shVEGFR2表达克隆转导HL60细胞后持续高水平抑制细胞。重组慢病毒Lenti6/shVEGFR2具有抑制白血病小鼠骨髓血管生成的作用。②VEGFR2 siRNA可阻断白血病HL60细胞VEGF/VEGFR2自分泌的内环路,在体外、动物体内均得到验证。③VEGF/VEGFR2自分泌链和旁分泌链的联合阻断加强了抗白血病效果。

BACKGROUND： Vascular endothelial growth factor receptor 2 （VEGFR2） is primarily involved in vascular endothelial growth factor （VEGF）-mediated signal transduction and plays a critical role in the pathological angiogenesis that occurs in a number of diseases, including leukemia. Besides, VEGF secreted by leukemia cells also induces its own expression which leads to an enhanced production of VEGFR2 which contributes to the survival and proliferation of leukemia cells. OBJECTIVE： To evaluate the inhibitive effect of Lenti6/shVEGFR2 on the VEGFR2 expression and leukemia growth in mouse. DESIGN： A randomized, parallelized, controlled and open tria SETTING： Department of Pediatrics, the Second Affiliated Hospital of Sun Yet-sen University; Biotechnology Research Center, Sun Yet-sen University, MATERIALS： The experiment had been done in the laboratories for Medical Research Center of the Second Affiliated Hospital, Sun Yet-sen University and Biotechnology Research Center, Sun Yet-sen University from May 2004 to January 2006. HL60, EA-hy926 cell line were used as the source of leukemia and human vascular endothelial cell. Block-iT Lentiviral RNAi Expression System was purchased from Invitrogen, Co.,Ltd.; human VEGFR2 Mcb （PE） was purchased from R＆D; CD31 immunohistochemistry kit was purchased from Boster, Co.,Ltd.; CD33-PE fluorescence labeled antibody was purchased from BD, Co.,Ltd. METHODS： ①The most efficient VEGFR2 small interfering RNA （siRNA） was designed and screened, then VEGFR2 short hairpin RNA （shRNA） was synthesized, ② The pU6/shVEGFR2 entry clone was constructed. Cells were transfected transiently and expression clone （Lenti6/shVEGFR2） was constructed, then cotransfected with ViraPowerTM Packaging Mix into 293FTTM cell. Lentiviral vectors harboring each Lenti6/shVEGFR2 were generated. ③ After transfecting with pU6/shVEGFR2 entry clone and transducting with Lenti6/shVEGFR2 expression clone, the effect on the development of leukemia was evaluated in an HL60 human leukemia cell line by mononuclear cell direct cytotoxicity assay. ④ In an HL60 intravenous xenograft leukemia mouse model, the distribution of microvessels in mouse bone marrow was observed after injecting human endothelial cells. ⑤ NOD/SCID mice were divided into 5 groups： leukemia model mouse （group A）; leukemia model mouse injected with recombinant lentivirus （group B）; leukemia model mouse injected with recombinant lentivirus and endothelial cell （group C）; leukemia model mouse injected with endothelial cell （group D）. Through detecting changes of CD33 positive cells and microvessel density （MVD） in bone marrow, observing peripheral blood cell （PBC） smear and slice of liver, spleen, the effect of Lenti6/shVEGFR2 recombinant lentivirus on mouse leukemia was evaluated. MAIN OUTCOME MEASURES： ① Assessment of VEGFR2 siRNA. ② Effect on expression of VEGFR2 after transfecting HL60 cell with pU6/shVEGFR2 entry vector. ③ Comparison of growth inhibitions between transfection with pU6/shVEGFR2 entry vector and transduction with Lenti6/shVEGFR2 expression vector in HL60 cell. ④Effect of shRNA mediated with lentivirus on VEGFNEGFR2 paracrine and autocrine loops in leukemia mouse, RESULTS： ① Effect on VEGFR2 expression after pU6/shVEGFR2 entry vector transfecting HL60： siRNAc was the most effective in inhibiting HL60 cell. pU6/shVEGFR2 entry clone constructed according to it had cell inhibitory rate as high as 84.9%. Expression of VEGFR2 mRNA and protein decreased significantly. ② Comparison of cell growth inhibitive rates after transfection of pU6/shVEGFR2 entry clone and transduction of Lenti6/shVEGFR2 expression clone： 48 hours after transfection of pU6/shVEGFR2 entry clone and transduction of Lenti6/shVEGFR2 expression clone, the cell growth inhibitive rates were similar. However, the cell growth inhibitive rate of entry clone descended rapidly after 48 hours （P 〈 0.01）; which of expression clone changed slowly, reaching the peak at 96 hours, dropped slightly, having no significance deviation. ③ Effect of shRNA mediated with lentivirus on VEGFNEGFR2 paracrine and autocrine loops in leukemia mouse： The amount of HL60 cells in bone marrow of groups A, B and C detected with flow cytometry were （25.8%± 4.9）%, （14.3%±5.1）%, （8.4±2.6）%, respectively （P 〈 0.05）; MVD in group C was obviously less than that in group D （P 〈 0.05）; The amount of HL60 cells in leukemia model mouse injected with recombinant lentivirus and endothelial cell was the lowest as compared with the other groups. CONCLUSION： pLenti6/shVEGFR2 expression clone transduces HL60 cell with lasting high level cell growth inhibition; Recombinant lentiviral Lenti6/shVEGFR2 can inhibit angiogenesis in bone marrow of leukemia mouse; VEGFR2 siRNA can block internal autocrine VEGFNEGFR2 loops, which has been validated in vivo and in vitro; combined blockage of VEGFNEGFR2 paracrine and autocrine loops strengthens anti-leukemia effect.