摘要
目的检测尤文肉瘤融合基因EWSFLI1和人粒单核细胞集落刺激因子(hGMCSF)基因修饰的树突细胞(DC)疫苗对尤文肉瘤细胞株A673的杀伤作用。方法利用hGMCSF腺病毒(AdhGMCSF),在1000U/mlIL4作用下,从外周血单个核细胞(PBMC)诱生树突细胞(DC)并对其表型进行流氏细胞仪(FCM)分析,通过同种混合淋巴细胞反应以检测其免疫刺激活性。在lipofectamine的介导下,将含有尤文肉瘤融合基因EWSFLI1的真核表达质粒pCA13/EWSFLI1转染DC,通过RTPCR检测EWSFLI1的表达,并通过乳酸脱氢酶(LDH)释放试验检测其对A673细胞的杀伤作用。结果应用AdhGMCSF从PBMC成功诱生出CD83、CD80、CD86及HLADR高表达,CD14低表达的DC,其对同种淋巴细胞有很强的免疫刺激活性。pCA13/EWSFLI1转染DC后,RTPCR检测DC中有EWSFLI1mRNA的表达。杀伤试验结果表明,AdhGMCSF诱生、EWSFLI1修饰的DC,体外诱导抗原特异的CTL,效靶比为20∶1时对A673细胞杀伤率为(29.9500±0.0117)%,高于对照组(P<0.01)。结论AdhGMCSF能够成功地从PBMC中诱生出有功能的DC。真核表达质粒pCA13/EWSFLI1在lipofectamine的介导下转染AdhGMCSF诱生的DC,能在DC中有效地表达。此EWSFLI1基因修饰的、AdhGMCSF诱生的DC体外致敏自体T淋巴细胞,生成抗原特异CTL。
Objective To Investigate the antitumor immunity of the EWS FLI1 gene and hGM CSF gene modified dendritic cells (DC) in vitro.Methods With the hGM CSF adenovirus (AdhGM CSF) and 1 000 U/ml IL 4,the DCs from peripheral mononuclear cells were cultured and analyzed by fluorescence activated cell sorting (FACS) and mixed lymphocyte reaction (MLR).Following lipofectamine mediated transfection to hGM CSF gene modified DC with expressing plasmid pCA13/EWS FLI1 encoding the EWS FLI1 gene,the expression of chimeric gene EWS FLI1 was detected by RT PCR.The antigen specific CTL was induced by DCs modified with the EWS FLI1 gene and hGM CSF gene.A LDH release assay was used to analyze the antigen specific CTL response against A673 cells.Results The DCs modified with hGM CSF gene showed the typical shape,high level expression of CD83,HLA DR,CD80,CD86 and acted as a potent immune stimulator to allogenic lymphocyte,which was similar to traditionally cultured DC.After the DC were transfected with pCA13/EWS FLI1,the EWS FLI1 mRNA was easily detected by RT PCR.The antigen specific CTL was induced successfully by the EWS FLI1 gene and hGM CSF gene modified DC.The vigorous antigen specific CTL response against A673 cells was detected by LDH release assay.The percent killing was (29.950?0±0.011?7)% at effector target ratio 20∶1,which was more efficient than that of the control.Conclusion The chimeric gene of Ewing sarcoma EWS FLI1 can be expressed in the DCs modified with hGM CSF gene after T lymphocytes were stimulated by DCs modified with EWS FLI1 gene and hGM CSF gene.The vigorous antigen specific CTL activity against A673 cells can be detected.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2004年第7期848-851,F003,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30000763)
关键词
尤文肉瘤
基因治疗
树突细胞
腺病毒
Ewing sarcoma
Gene therapy
Dendritic cell
Adenovirus
