Inhibition of leukemia mediated by vascular endothelial growth factor receptor 2 short hairpin RNA gene silencing / 中国组织工程研究

ABSTRACT

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.OBJECTrVE To evaluate the inhibitive effect of Lenti6/shVEGFR2 on the VEGFR2 expression and leukemia growth in mouse.DESIGN: A randomized, parallelized, controlled and open trial.SETTING: Department of Pediatrics, the Second Affiliated Hospital of Sun Yat-sen University; Biotechnology Research Center, Sun Yat-sen University.MATERIALS The experiment had been done in the laboratories for Medical Research Center of the Second Affiliated Hospital, Sun Yat-sen University and Biotechnology Research Center, Sun Yat-sen University from May 2004 to January 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.transiently and expression clone (Lenti6/shVEGFR2) was constructed, then cotransfected with ViraPowerTM Packaging Mix pU6/shVEGFR2 entry clone and transducting with Lenti6/shVEGFR2 expression clone, the effect on the development of intravenous xenograft leukemia mouse model, the distribution of microvessels in mouse bone marrow was observed after 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.mediated with lentivirus on VEGFNEGFR2 paracrine and autocrine loops in leukemia mouse.effective in inhibiting HL60 cell. pU6/shVEGFR2 entry clone constructed according to it had cell inhibitory rate as high as 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 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.