摘要
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 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 pheno- types of CTLs were analyzed by flow cytometry. T cell proliferation was judged by 3 H-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-euhured with RNA-transfected DCs got a significant increase of the CD8^+ expres- sion (25.58±2. 73)% higer than those of PBS-transfeeted DCs (26. 10±1.76)% and non-transfected DCs (25.58 ±2.73 ) % ( P 〈 0. 05 ). 2) RNA-transfeeted DCs can significantly stimulate autologous T cell proliferation. The counts per minute(CPM) of positive control, total RNA-transfeeted 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 construeted in nude mice. Transplanted tumor size of the RNA- transfected DCs group was (540 ±99 )mm^3 at the end of experiments and was significantly inhibited compared with the PBS-transfected DCs ( 1 572 ±147) mm^3 and non-transfected DCs(2 043±395)mm^3 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 ceils transfected with total RNA of lung adenocareinoma cell can stimulate antigen specific CTLs, which inhibited the growth of lung adenocarcinoma transplanted tumors in nude mice,resultlng in a statistical siguifleant differenee in comparing with the control groups.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2012年第4期312-317,共6页
Chinese Journal of Blood Transfusion
基金
国家自然科学基金面上项目(81172172)
安徽省卫生厅医学科研课题(09A082)
关键词
树突状细胞
细胞毒T淋巴细胞
肺腺癌
RNA
转染
抗肿瘤效应
裸鼠
Dendritic cells
Cytotoxic T lymphocytes
Lung adenocarcinoma
RNA
transfect
Anti-tumor effect
Nude mice