Anti-tumor Effect Of The Antigen Speciifc CTL Induced By Dendritic Cells Transfected With Total Rna Of Lung Adenocarcinoma Cell In Vivo

Objectives：Considering the difficulties of lung adenocarcinoma in absence of knowntumor antigens and the sources of tumor tissue in patients with advanced tumors, wetake advantages of tumor cell lines and tumor cells of living tissues sharing tumorantigens and built RNA-DCs vaccine to explore anti-tumor effects of the antigenspecific cytotoxic T lymphocytes (CTL) induced by RNA-DCs vaccine on tumor-bearing nude mice model.Methods：(1)DCs induced from the peripheral blood mononuclear cells were transfected with total RNA extracted from human lung adenocarcinoma A549cellsthrough electroporation and co-cultured with T lymphocyte cells to activate antigenspecific CTL(RNA-DCs-CTL).(2)DCs were divided into three groups: total RNA-transfected DCs(RNA-DCs), PBS-transfected DCs(PBS-DCs) and non-transfectedDCs(N-DCs). The proportions of CD8+T cells of different groups after mixedlymphocyte cultured were analyzed by flow cytometry. RNA transfection efficiencywas verified by RT-PCR.T cell proliferation was judged by3H-TdR incorporation assay.And The specific cytotoxicity was measured by lactate dehydrogenase(LDH)content.(3)Establish nude mice model by lung A549cell line. Tumor-loaded nude micewere inoculated with stimulated T cells and tumor volume and inhibitive rates of the3groups were compared. The expressions of Bax、Bcl-2、COX-2and VEGF in tumortissues were detected by immunohistochemistry and their mean optical density(MOD)were analyzed by using automatic image analysis system.Results：(1)The CTL by autologous T cells co-cultured with RNA-DCs got a significantincrease of the CD8+expression (P<0.05).(2)The A549total RNA can be transfectedthrough electroporation successfully and DCs can express the tumor antigens.(3)RNA-DCs can stimulate autologous T cells to proliferate effectively. AndRNA-DCs-CTL showed stronger selective in vitro cytotoxicity against the target tumorcells as compared with other groups(P<0.05).The counts per minute(CPM) of positivecontrol、 RNA-DCs、 PBS-DCs and N-DCs groups were17105±130,7759±493,2611±161,2248±332respectively(P<0.05).(4)The lung model was successfullyconstructed on nude mice. Transplanted tumor volume of the RNA-DCs group weresignificantly inhibited (P<0.05) compared with the PBS-DCs and N-DCs groups.(5)Theinhibitive rates of RNA-DCs and PBS-DCs groups were68.53%and8.62%respectively.(6)Immumohistochemistry displayed that the expression of Bax wasup-regulated, and the expressions of COX-2、Bcl-2and VEGF were down-regulated in the RNA-DCs group.Conclusion：(1)Dendritic cells transfected with total RNA of human lungadenocarcinoma A549cells can stimulate antigen specific CTL, which inhibited thegrowth of tumor-bearing nude mice, resulting in a statistical significant difference incomparing with the control groups.(2) In addition to the cytotoxic effects, anti-tumoreffects of A549-RNA-DCs-CTL may be associated with induction of tumor cellapoptosis and inhibition of tumor angiogenesis.