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Anti-tumor Effect of Dendritic Cell Vaccines Transfected with Total RNA of Human Lung Adenocarcinoma Cell Through Electroporation in Vitro

Author: JinJuan
Tutor: WangBaoLong
School: Anhui Medical University,
Course: Clinical Laboratory Science
Keywords: Human lung adenocarcinoma A549 RNA Dendritic cells Electric transfected
CLC: R734.2
Type: Master's thesis
Year: 2011
Downloads: 13
Quote: 0
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Abstract


Purpose: recombinant human granulocyte colony stimulating factor (human recombination granulocyte colony stimulating factor, rhG-CSF) mobilized peripheral blood stem cell collection was separation of dendritic cells (dendritic cells DCs), to explore the human lung adenocarcinoma cells total RNA electroporation line A549 transfected DCs to prepare the feasibility of the vaccine, and observe DCs vaccine in vitro induction of specific anti-tumor immune response capabilities. Methods: recombinant human granulocyte macrophage cells set off the stimulus factor (rhGM-CSF) and recombinant human interleukin-4 (rh IL-4) from the blood cell separator separating out the peripheral blood collected material induced by DCs produce, at the same time from people A549 cells in the total RNA was extracted for transfection of DCs after transfection of DCs co-cultured with autologous T cells induced to become cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL). Were divided not transfected DCs group, PBS, transfected DCs group, group of DCs transfected with RNA, flow cytometry (FCM) identification of DCs, T cell phenotype, RT-PCR proof of total RNA transfection efficiency, MTT detection T-cell proliferative capacity and lactate dehydrogenase (LDH) release assay determination of anti-tumor activity of the enzyme-linked immunosorbent assay (ELISA) to detect IL-12, IFN-γ secretion levels and compare the difference. Results: 1) by RT-PCR proved electroporation method can successfully make the A549 total RNA was transfected into DCs can DCS expressing tumor antigen. 2) RNA transfection group DCs surface markers CD40, CD80, CCR-7, CD83, HLA-DR were tested strongly positive expression, the expression rate significantly higher than the PBS the transfection group of DCs and untransfected group, the difference was statistically Significance (P lt; 0.05); 3) streaming results show significantly increased the proportion of RNA-transfected DCs after stimulation of T cell surface markers CD8; 4) RNA transfection group of IL-12, IFN-γ secretion levels significantly higher than that PBS transfection group and untransfected group (P lt; 0.05); 5) RNA transfection group DCs can effectively stimulate the proliferation of T lymphocytes and induction of tumor-specific CTLs, specific cytotoxicity of A549 cells. PBS transfected group and untransfected group no significant effect (P lt; 0.05). Conclusion: The use of density gradient centrifugation can after rhG-CSF mobilization of outside weeks dry blood cells collected material separation induced mobilization of peripheral blood stem cells collected material separation induced by the DCs have the typical morphology and immune table type of, the use of electric transfection method can be successfully transferred to the total RNA of A549 in DCs and their expression within the tumor antigen in vitro can induce tumor-specific immune response.

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CLC: > Medicine, health > Oncology > Respiratory system tumors > Lung tumors
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