Experimental Study On The Anti-tumour Effects Induced By Fusions Of DC With NCI-H460 Lung Cancer Cell Line Transfected With Interleukin 18

DC are the most powerful of all antigen -presenting cells and play a critical role in the induction of primary immune responses.DC-based vaccination represents a potentially powerful strategy for cancer imniunotherapy.Various approaches were established to construt tumour vaccines basing on DC,cytokine genes modify DC,Ag genes transfect DC,DC pulsed by mRNA,antigen peptides or cell lysate derived from tumour cells,fuse DC with tumour cells. Among which DC-based hybrid tumour vaccine seems to be an more attractive strategy for cancer immunotherapy.IL-18 is a pleiotropic functional cytokine with obvious antitumor effects. Combining IL-18 gene-modification with DC-based vaccines may lead to potentially increased therapeutic antitumor efficacy.Aimed at obtaining more powerful DC-based vaccines by combining tumor antigens with the antigen-presenting function of DC and immune promoting effects of IL-18,this study was conducted.In this study,we construct hybrid cDNA of IL-18 containing the signal sequence of IL-12 P40 subunit,transfect NCI-H460 cell line with this hybrid cDNA,then fuse DC with these gene-modified NCI-H460 cells. Antitumor effects induced by these fusions are to be analysed , taking Ag-pulsed DC and non-modified fusions as control. Through which todetermine if IL-18 gene modified fusions are more effective than Ag-pulsed DC andnon-modified fusions of DC with NCI-H460 cells in inducing antitumor immunity.This study consists of five parts as follows: Fart 1 Induction and identification of DCAdherent mononuclear cells were separated from human peripheral blood and monocytes were abtained by wall-adherent method from PBMC.Monocytes were cultured with RPMI-1640 containing GM-CSF and IL-4.On the 5th day ,TNF- a was added to the culture system to induce DC maturation.DC were observed with light microsope .DC were harvested On the 8th day and ultrastructure and phenotypic analysis were performed with electron microscope and Flow Cytometry respectively.MTT method was used to determine the proliferation of T cells stimulated by DC on the 8th day.After 8 days culture with cytokines ,monocytes developed into DC, which revealed typical morphological dendrites, high level of costimulatory molecules and significant stimulating function on T cells proliferation.It was proved that we induced mature DC with bioactivity from monocytes. Part 2 Construction of recombinant plasmid containing secreted human IL-18PBMC stimulated with LPS were harvested and from which RNA was extracted with Trizol.RT-PCR was conducted to clone IL-12 P40 signal sequence and mature IL-18 sequence.Hybrid cDNA of these 2 fragments was acquired by overlap extention PCR.Connections of hybid cDNA and pGEM-T vector were transduced into E.coli strain JM109.Plasmids were extracted from positive clone propogation,and the recombinant plasmids were identified by enzyme maping.After sequencing,hybrid cDNA were inserted into pcDNA3.1+ expression vector,then transduction ,propogation and restriction endonuclease digestion steps were the same as mentioned above. Enzyme maping showed the inserted fragment of 548bp in length was cosistant with the supposed length. Sequencing confirmed that the hybrid fragment was in conformity with the sequences of IL-12 P40 and mature IL-18 from Genbank.In conclusion,we obtained hybrid cDNA consisting of IL-12 P40 signal sequence and human IL-18 mature peptide cDNA and constructed secreted IL-18 expression recombinant plasmid successfully.Part 3 Eukaryotic expression of secreted human IL-18NCI-H460 cells were cultured routinely. Cells in optimal physiological condition were collected and transfected with secreted IL-18 recombinant plasmid by positive liposomes .When G418-resistant clones formed after cultured in G418 culture system,we sucked clones and selecting cultured these clones in G418 for 3-4 weeks.RT-PCR analysis was performed to investingate the mRNA expression of hybrid IL-18 cDNA in hybrid-gene-transfected cells and G418-resistant genes in pcDNA3.1+- transfected cells.Western blot analysis w...