口服mHags，AML/ETO菌蜕疫苗抗白血病的研究

项目名称： 口服mHags，AML/ETO菌蜕疫苗抗白血病的研究

项目编号： No.30800486

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 胡海燕

作者单位： 南方医科大学

项目金额： 20万元

中文摘要： 本课题1）成功构建了AML/ETO基因阳性的白血病裸鼠模型。2）课题原计划将HA-1、2,HY和AML/ETO质粒，插入大肠杆菌菌蜕， 但多基因质粒稳定性差，故仅选择表达最广泛的HA-1全长或优势片段与AML/ETO构建双基因质粒，另因大肠杆菌菌蜕包裹效率不稳定免疫反应过于强烈，改为减毒沙门氏菌作为递送体感染白血病细胞Kasumi-1细胞，活化NK细胞。透射电镜证实沙门氏菌为胞内寄生菌，流式细胞检测Kasumi-1细胞高表达NK细胞活化配体ULBP2，但低表达MICA/B和ULBP1，转染单基因、双基因质粒后，ULBP1及MICA/B有显著提高，以沙门氏菌递呈的HA-1优势片段联合AML/ETO基因的双质粒感染组，3种配体表达均超过90%，LDH释放试验结果显示，3例正常人外周血NK细胞对感染了携带外源质粒沙门氏菌的Ksaumi-1细胞杀伤效应显著高于对照组；ELISA试验显示与对照组相比，NK细胞分泌IFN-γ27700;平显著提高，同时测各组共培养后的NK细胞miR-155，miR-106b及miR-92a水平，试验组显著提高。3）证实原癌基因miR-21在白血病耐药及增殖起重要作用。

中文关键词： ML/ETO融合基因；次要组织相容抗原HA-1；自然杀伤细胞；miR；白血病裸鼠模型

英文摘要： 1)We have established the AML/ETO positive leukemia model on nude mice.2)The initial project planned to pack HA-1、2,HY and AML/ETO plasmid by E.coli bacteria ghost. Due to the instability of multi-gene plasmid, we changed to recombine AML/ETO with HA-1 dominant antigen peptide coding sequence or full-length HA-1, which gene is widely expressed in leukemia cells. Besides, considering the instability and over immune reaction of E.coli ghost, the attenuated Salmonella was adopted as the carrier of recombinant plasmids to acute myeloblastic leukemia cell Kasumi-1. We expect the infected kasumi-1 cells will activate immunity of NK cells.Transmission Electron Microscope was used to prove the intracellular residency of Salmonella; Flow Cytometry results showed the high-expression of NK cells activation ligands ULBP2, while low-expression of ULBP1 and MICA/B;Infected with single-gene or double-gene plasmid,ULBP1 and ULBP2 ligand level obviously enhanced. Transfected with HA-1 dominant antigen peptide coding sequence and AML/ETO fusion gene dual plasmid, the kasumi-1 cells activated MICA/B, ULBP1 and ULBP2 ligands on NK cells, with expression over 90%. LDH release assay showed NK cells from 3 healthy individuals' peripheral blood have more intense effects on Kasumi-1 cells, infected by recombinant Salmonella, than other groups. In addition, IFN-γecreted by NK cells in test group largely exceeded that of the control group in an ELISA assay. Finally, miR-155，miR-106b and miR-92a level presented an increase in NK cells, which were treated with infected leukemia cells. 3）Our results further proves oncogene miR-21 plays a significant role in drug resistance and proliferation of leukemia.

英文关键词： AML/ETO fusion gene；minor histocompatibility antigen HA-1；Natural killer cell；microRNA；leukemia nude mouse model