论文部分内容阅读
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)%(P<0.05);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(P<0.05);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(P<0.05);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。
Objective To investigate the anti-tumor effects of antigen-specific cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transfected with total RNA of human lung adenocarcinoma cells in nude mice. Methods 1) DCs were induced from peripheral blood mononuclear cells (PBMCs) and total RNA was extracted from human lung adenocarcinoma A549 cells. The transfected DCs were mixed with autologous T cells to induce antigen - specific CTLs. 2) The experiment was divided into DCs transfected DCs group, DCs transfected PBS group and non-transfected DCs group, flow cytometry (FCM) identified T cell phenotype, 3H-TdR incorporation of T cell proliferation; 3 ) To establish a lung cancer cell line A549 tumor-bearing nude mice model, adoptively transfused CTLs, tumor volume comparison to calculate the tumor inhibition rate, immunohistochemistry to detect the expression of Bax, Bcl-2, COX-2 and VEGF in tumor tissue Automatic image analysis system to analyze the average optical density (MOD) value. Results (1) CD8 + T cells were significantly up-regulated in CTLs transfected with transfected DCs and autologous T cells (79.29 ± 2.18)%, which was significantly higher than that in CD8 + T cells transfected with DCs (26.10 ± (P <0.05); 2) DCs transfected with RNA significantly stimulated the proliferation of autologous T cells, and the cpm values obtained by 3H-TdR infiltration were positive 17 105 ± 130 in the control group, 7 759 ± 493 in the DCs transfected with RNA, 2 611 ± 161 in the DCs transfected with PBS and 2 248 ± 332 in the untransfected DCs group (P <0.05); 3) At the end of the experiment, the tumor-bearing volume (540 ± 99) mm3 in nude mice transfected with RNA was significantly lower than that in transfected PBS (1572 ± 147) mm3 and non-transfected 395) mm3 (P <0.05). 4) The inhibition rate of transfected RNA group and transfection PBS group were 68.53% and 8.62%, respectively; 5) CTLs induced by transfected RNA DCs could significantly up-regulate the expression of Bax The MOD value of the stained RNA group was 335 ± 105, and the expressions of Bcl-2, COX-2 and VEGF were significantly down-regulated (MOD values of 146 ± 47, 122 ± 33 and 128 ± 58, respectively) Conclusion The total RNA of human lung adenocarcinoma cells transfected DCs induced antigen-specific CTLs on lung adenocarcinoma xenografts in nude mice growth inhibition.