腺病毒介导人FL与GM-CSF联合抗肿瘤免疫基因治疗实验研究  

Adenovirus mediated human FL and GM-CSF combinational antitumor immunogenetherapy

作　　者：张艳丽 魏义举 王亚栋 邓艳春 苏林 刘长征 杨克恭 陈松森 

机构地区：[1]中国医学科学院医学分子生物学国家重点实验室中国医学科学院基础医学研究所中国协和医科大学基础医学院,北京100005

出　　处：《中国免疫学杂志》2006年第7期623-628,共6页Chinese Journal of Immunology

基　　金："十五"国家高技术"863"计划资助项目(2001AA212171)

摘　　要：目的:研究腺病毒介导人F lt3 ligand(hFL)与GM-CSF在体内外的表达、体外生物学活性,体内扩增小鼠脾脏DCs及抑制小鼠EL-4淋巴瘤的效应。方法:应用AdEasy系统,将hFL和hGM-CSF DNA重组到穿梭质粒的两个独立转录单位上,得到重组腺病毒Ad-hFL-hGM-CSF。采用ELISA、W estern b lot法测定重组腺病毒介导的hFL和hGM-CSF在体内外的表达情况。MTT法测定hFL和hGM-CSF刺激细胞增殖的生物学活性。利用流式细胞分析技术研究重组腺病毒对小鼠脾脏树突状细胞(DCs)的扩增情况。在移植EL-4淋巴瘤的小鼠皮下注射4次重组腺病毒(每次5×109pfu),研究其抑制肿瘤生长的功能。结果:构建得到含有双基因的重组腺病毒Ad-hFL-hGM-CSF,并在体内、外高效共表达hFL和hGM-CSF。hFL对小鼠骨髓有核细胞,hGM-CSF对TF1细胞,均有明显的刺激增殖作用。Ad-hFL-hGM-CSF显著扩增小鼠脾脏DCs至正常值的50倍。抑瘤实验中,Ad-hFL-hGM-CSF使小鼠EL-4淋巴瘤生长延慢,瘤体比对照组明显减小,抑瘤率为30%,P<0.05。病理切片显示,Ad-hFL-hGM-CSF治疗组肿瘤细胞间隙有大量淋巴细胞浸润。结论:腺病毒介导hFL和hGM-CSF在小鼠体内显著扩增脾脏DCs,并可抑制EL-4淋巴瘤的生长。Objective ：To investigate the expression of human Flt3 Ligand（hFL） and human GM-CSF（hGM-CSF） mediated by adenovirus vector, its biological activity in vitro, DCs expansion function and antitumor effect in vivo. Methods：The recombinant adenovirus Ad-hFL-hGM-CSF was constructed based on the AdEasy system, with hFL and hGM-CSF DNA respectively cloned into two independent transcriptional unit. HEK 293 cells were infected by the recombinant adenovirus to test the expression of hFL and hGM-CSF in vitro. The expression of hFL and hGM-CSF were evaluated by ELISA and Western blot. The cell proliferation effects of hFL and hGM-CSF were evaluated by MTT assay. In vivo DCs expansion effects of Ad-hFL-hGM-CSF were tested using FACS. In the antitumor test, recombinant adenovirns Ad-hFL-hGM-CSF（5 × 10^9 pfu） was injected subcutaneously in mice with EIA lymphoma for four times. The tumor growth was measured every 2 or 3 days. Results：The recombinant adenovirns Ad-hFL-hGM-CSF expressed hFL and hGM- CSF effectively in vitro and in vivo. The expressed hFL notably increased the cell number of bone mann-ow nucleated cells cultured in vitro and the expressed hGM-CSF can stimulate the proliferation of TF1 cells. DC number in the spleen of Ad-hFL-hGM-CSF treated mice markedly increased to five decuple of original. In the antitumor test Ad-hFL-hGM-CSF inhibited the growth of the EL4 lymphoma in mice with a rate of about 30%, P 〈 0. 05. Conclusion：Adenovirus can mediate efficient expression of hFL and hGM-CSF which expand DCs dramatically in vivo, and have antitumor activity towards EL-4 lymphoma.

关 键 词：HFL 树突状细胞(DC) 腺病毒 免疫基因治疗 

分 类 号：Q782[生物学—分子生物学]