foxp3转染的CD4^+ CD25^- T细胞治疗急性肺损伤的研究  

Therapeutic study on the actue lung injury by adoptive transfer of gene foxp3-transfected CD4^+ CD25^- T lymphocytes

作　　者：张方[1] 施毅[1] 李子玲[1] 胡波[2] 宋勇[1] 

机构地区：[1]南京军区南京总医院呼吸内科 [2]南京军区联勤部卫生部,南京210002

出　　处：《中国组织化学与细胞化学杂志》2011年第1期17-22,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：中国博士后科学基金资助(20070411050);江苏省博士后科研资助计划(0701023B);南京军区面上课题(07M074)

摘　　要：目的通过基因转染技术将foxp3基因导入CD4+CD25-T淋巴细胞,生成CD4+CD25-Foxp3+T细胞,通过输入急性肺损伤小鼠模型体内,促进炎症消退,为体外定向操纵调节T细胞生成和细胞过继治疗急性肺损伤奠定基础。方法采用RT-PCR法从小鼠胸腺来源cDNA文库中扩增获得foxp3 cDNA,亚克隆至构建pIRES2-EGFP质粒中,构建并筛选出重组质粒pmFoxp3-IRES2-EGFP;采用免疫磁珠法分离CD4+CD25-T淋巴细胞,穿孔法转染Foxp3基因至CD4+CD25-T淋巴细胞,流式细胞仪检测CD4+和EGFP共表达的细胞比例;将foxp3基因转染的小鼠CD4+CD25-T细胞通过尾静脉输注急性小鼠体内,通过ELISA法检测小鼠肺泡灌洗液中TNF-α以及IL-1β细胞因子的表达,通过肺组织病理研究肺部炎症的消退状况。结果成功构建双基因共表达重组质粒pmFoxp3-IRES2-EGFP;免疫磁珠法较高纯度分离CD4+CD25+、CD4+CD25-T淋巴细胞,电穿孔法成功将重组质粒pmFoxp3-IRES2-EGFP转染致CD4+CD25-T细胞,CD4+和EGFP共表达的细胞比例为35.5%;过继Foxp3基因转染的小鼠CD4+CD25-T细胞以及Treg均能抑制急性肺损伤小鼠肺泡灌洗液中TNF-α以及IL-1β炎性细胞因子的表达,促进炎症的消退。结论转染Foxp3基因的CD4+CD25-细胞同Treg(CD4+CD25-)细胞一样可以通过细胞过继治疗急性肺损伤。Objective To transfer the foxp3 gene into CD25-CD4＋ T lymphocytes to induce the production of CD25-CD4＋ Foxp3＋ T lymphocytes,then infuse it to the mouse model of acute lung injury to promote the subsidence of inflammation.It is a novel way to treat acute lung injury by guiding T regulatory lymphocytes in vitro.Methods The foxp3 cDNA was amplified from Balb/c mouse monocyte suspension by RT-PCR,and subcloned into pIRES2-EGFP plasmids to construct the recombinant plasmids pmFoxp3-IRES2-EGFP.CD4＋ CD25-T lymphocytes were separated with immunomagnetic beads,and the foxp3 gene was transfected into CD4＋ CD25-T lymphocytes with electroporation.The ratio of EGFP and CD4＋ coexpression was determined by flow cytometry.After the foxp3 gene transfected CD4＋ CD25-T lymphocytes were injected into the mouse model of acute lung injury via vena caudalis,the concentrations of TNF-α and IL-1β in BALF were measured by enzyme linked immunosorbent assay（ELISA）,and the histological changes of lung inflammation were examined by light microscopy.Results Eukaryotic coexpression plasmids pmFoxp3-IRES2-EGFP were constructed successfully,and highly purified CD4＋ CD25-and CD4＋ CD25-T lymphocytes were separated with immunomagnetic beads.After electroporation with pmFoxp3-IRES2-EGFP,the ratio of cells with EGFP and CD4＋ coexpression was 35.5%.The concentrations of TNF-α and IL-1β in BALF were decreased significantly,and the lung inflammation subsided after the adoptive transfer of foxp3 gene transfected CD4＋ CD25-T lymphocytes or CD4＋ CD25-T lymphocytes.Conclusion The foxp3 gene transfected mouse CD4＋ CD25-T lymphocytes may promote the subside of lung inflammation as CD4＋ CD25-T lymphocytes,and may play an important role in treating acute lung injury by adoptive transfer.

关 键 词：急性肺损伤 FOXP3 基因治疗 TREG 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学]