肺腺癌细胞总RNA转染树突状细胞诱导特异性CTLs的体内抗肿瘤效应研究

目的探讨人肺腺癌细胞总RNA转染的树突状细胞(DCs)诱导抗原特异性的细胞毒T细胞(CTLs)对移植瘤裸鼠的抗肿瘤作用。方法 1)从外周血单个核细胞(PBMCs)中诱导DCs,提取人肺癌细胞A549总RNA,用于电转染DCs,转染后的DCs与自体T细胞混合培养,诱导抗原特异性的CTLs;2)实验分为转染RNA的DCs组、转染PBS的DCs组和未转染DCs组,流式细胞术(FCM)鉴定T细胞表型,3H-TdR掺入检测T细胞增殖能力;3)建立肺癌细胞株A549荷瘤裸鼠模型,过继回输CTLs,比较肿瘤体积,计算抑瘤率,免疫组化法检测移植瘤组织中Bax、Bcl-2、COX-2和VEGF的表达,并用全自动图像分析系统分析其平均光密度值(MOD)。结果 1)转染RNA的DCs和自体T细胞共育诱导的CTLs的CD8+T细胞大幅度上调,表达率(79.29±2.18)%明显高于转染PBS的DCs组CD8+T细胞(26.10±1.76)%和未转染DCs组CD8+T细胞(25.58±2.73)%(P0.05);2)转染RNA的DCs显著刺激了自体T细胞增殖,3H-TdR渗入所得的cpm值:阳性对照组为17 105±130,转染RNA的DCs组为7 759±493,转染PBS的DCs组为2 611±161,未转染DCs组为2 248±332(P0.05);3)成功建立肺癌裸鼠移植瘤模型,实验结束时,转染RNA组裸鼠荷瘤体积(540±99)mm3明显小于转染PBS组(1 572±147)mm3和未转染组(2 043±395)mm3(P0.05);4)转染RNA组和转染PBS组的抑瘤率分别为68.53%和8.62%;5)转染RNA的DCs诱导的CTLs能显著上调Bax的表达(转染RNA组MOD值为335±105),显著下调Bcl-2、COX-2和VEGF的表达(转染RNA组MOD值分别为146±47、122±33和128±58)。结论人肺腺癌细胞总RNA转染DCs诱导的抗原特异性CTLs对肺腺癌裸鼠移植瘤的生长产生抑制作用。

Anti-tumor effect of the antigen specific CTLs induced by dendritic cells transfected with total RNA of lung adenocarcinoma cell in vivo

Objective Explore anti-tumor effect of the antigen specific cytotoxic T lymphocytes(CTLs) induced by dendritic cells(DCs) transfected with total RNA of lung adenocarcinoma cell in nude mice bearing lung A549 cell line.Methods 1)DCs induced from the peripheral blood mononuclear cells were transfected with total RNA extracted from lung adenocarcinoma A549 cells through electroporation,and co-cultured with T cells to activate antigen specific CTLs.2)DCs were divided into three groups:total RNA-transfected DCs,PBS-transfected DCs and non-transfected DCs.The phenotypes of CTLs were analyzed by flow cytometry.T cell proliferation was judged by 3H-TdR incorporation assay.3)Establish nude mice model by lung A549 cell line.Tumor-loaded nude mice were inoculated with CTLs and tumor sizes and inhibitive rates of the 3 groups were compared.The expressions of Bax、Bcl-2、COX-2 and VEGF in tumor tissues were detected by immunohistochemistry and their mean optical density(MOD) were analyzed by using automatic image analysis system.Results 1)The CTLs by autologous T cells co-cultured with RNA-transfected DCs got a significant increase of the CD8+ expression(25.58±2.73)% higer than those of PBS-transfected DCs(26.10±1.76)% and non-transfected DCs(25.58± 2.73)%(P<0.05).2)RNA-transfected DCs can significantly stimulate autologous T cell proliferation.The counts per minute(CPM) of positive control、total RNA-transfected DCs、PBS-transfected DCs and non-transfected DCs groups were 17 105±130,7 759±493,2 611±161,2 248±332 respectively(P<0.05).3) The lung model was successfully constructed in nude mice.Transplanted tumor size of the RNA-transfected DCs group was(540±99)mm3 at the end of experiments and was significantly inhibited compared with the PBS-transfected DCs(1 572±147) mm3 and non-transfected DCs(2 043±395)mm3 groups(P<0.05).4)The inhibitive rates of RNA-transfected DCs and PBS-transfected DCs groups were 68.53% and 8.62% respectively.5) It was displayed that the expression of Bax was up-regulated(the MOD of RNA-transfected DCs group was 335±105),and the expressions of COX-2、Bcl-2 and VEGF were down-regulated in the RNA-transfected DCs group(the MODs were 146±47、122±33 and 128±58 respectively).Conclusion DC cells transfected with total RNA of lung adenocarcinoma cell can stimulate antigen specific CTLs,which inhibited the growth of lung adenocarcinoma transplanted tumors in nude mice,resulting in a statistical significant difference in comparing with the control groups.