负载LMP-1基因的树突状细胞诱导CTL对鼻咽癌细胞的杀伤效应  

The Killing Effect of CTL Induced by Dendritic Cells Transfected with LMP-1 Gene on Nasopharyngeal Carcinoma Cell

作　　者：徐瑞凤[1] 石敏[1] 尤长宣[1] 吕成伟[1] 罗荣城[1] 廖旺军[1] 

机构地区：[1]南方医科大学南方医院肿瘤中心,广东广州510515

出　　处：《河北医学》2010年第12期1409-1413,共5页Hebei Medicine

基　　金：2007年广东省科技计划项目(2007B030704012);2008年广州市科技计划项目(2008J-C131)

摘　　要：目的:探讨超声联合微泡造影剂能否增强绿色荧光蛋白(GFP)质粒pEGFP-C3-LMP-1转染树突状细胞(Dendritic cells,DC),进而探讨转染LMP-1基因的DC诱导细胞毒性T淋巴细胞(CTL)对鼻咽癌(NPC)细胞株的体外杀伤作用。方法:体外培养的人DC分为四组:①单纯质粒组(A组):加入质粒;②超声照射组(B组):质粒+超声辐照;③微泡造影剂组(C组):质粒+微泡造影剂;④超声+微泡造影剂组(D组):质粒+微泡造影剂+超声辐照。荧光显微镜观察GFP质粒在DC内的表达,流式细胞仪评价GFP质粒的转染效率。另将体外培养的DC分为两组用于检测DC表面表型的表达及体外杀伤实验:①空白对照组,单纯培养的DC;②基因转染组,具体实验方法同上述D组。流式细胞仪检测DC表面分子表型的表达。DC和T细胞混合共培养获得的CTL为效应细胞,LMP-1表达阳性的NPC细胞株C666-1为靶细胞,按效靶比10:1的比例混合培养24h后,MTT法检测CTL对靶细胞的杀伤作用。结果:D组DC内有较多的绿色荧光表达,且转染效率最高(14.86±2.36)%,和其余各组均有显著性差异。LMP-1基因转染组DC表面标志CD80、CD83、CD86、CD40、HLA-DR的表达明显高于对照组,所诱导产生CTL对靶细胞有明显的杀伤作用,其杀伤效率为(41.72±3.53)%,明显高于空白对照组(7.62±2.36)%。结论:超声联合微泡造影剂可有效地介导GFP质粒转染DC。LMP-1基因转染组DC诱导产生特异性的CTL对靶细胞有明显的杀伤作用。Objective： To explore whether ultrasound exposure combined with microbubble destruction could be used to enhance the delivery efficiency of GFP plasmid pEGFP--C3--LMP-1 into dendritic cells （DC） and the anti--tumor effect of cytotoxic T lymphocytes （CTL） induced by DC in vitro. Method： DC were cultured in vitro and divided into 4 groups： Group A, plasmid alone; Group B, plasmid and ultrasound irradiation; Group C, plasmid and microbubble; GroupD, plasmid with ultrasound irradiation and microbubble. Transfection rate of the transferred gene was assessed by fluorescent microscopy and FACS analysis. In addition, DC were divided into 2 groups to detect the biomarkers of DC and study the killing effect in vitro： （1）blank control group,the cultured DC only ; （2）gene transferring group,specific experiment method as aforementioned group D. The biomarkers of dendritic cells were assessed by FACS analysis. The CTL were obtained by co--culturing DC and T cells, and then the CTL and LMP1 positive nasopharyngeal carcinoma cell line C666-1 were mixed at the ratio of 10：1 for 24h. The cytotoxicity of the CTL was tested by MTT assay. Result： .There were significant differences between group D and other groups in transfection efficiency. The transfection efficiency of group D was the highest up to （14.86 ±2.36）%. Compared with other groups, the gene transferring group expressed higher level of biomarkers, including CD80, CD83, CD86, CD40, HLA--DR. The killing efficiency of CTL induced by DC of gene transferring group was[41. 72±3.53）%, which was significantly higher than that of blank control group. Conclusion： Ultrasound combined with microbubble destruction could induce effective transfection of pEGFP- C3--LMP-1 gene into DC. The CTL induced by DC which was transfected by pEGFP--C3--LMP -1, had a specific killing effect on LMP1 positive nasopharyngeal carcinoma cell line C666-1.

关 键 词：超声微泡造影剂 树突状细胞 基因转染 鼻咽癌 免疫治疗 

分 类 号：R739.63[医药卫生—肿瘤]