Immunotherapy Research On Pancreatic Cancer Induced By Dendritic Cells Pulsed With Mutant K-ras Peptide

The prognosis for pancreatic cancer patient is consistently poor because its anatomical and biological character. Most patient with pancreatic cancer died of local recurrence or metastatic spread,thus new therapeutic approaches are needed to improve the outcome of pancreatic cancer.Overwhelmmg evidences have demonstrated that dendritic cell(DC),the most potent professional antigen presenting cell(APC),is efficient in phagocytosing antigens and presenting antigens to native T cells.The mature DC can stimulate and activate native T cells and play an important role in initiating innate and adaptive immune responses,especially the potent antitumor immunity.Recently,how to induce the special antitumor immunity by DC vaccine loaded with tumor associated antigen(TAA)/tumor special antigen(TSA),has become problem urgent to be solved.K-ras is a proto-oncogene that is mutanted in 75-100% of pancreatic cancers,its mutant type is point mutation,which take place in 12, 13, 61 codon. Having detected K-ras mutant ion by RT-PCR and direct sequencing methods,We produced k-ras DC vaccine by pulsed with synthesized mutant peptide.To provide the theoretical and experimental evidence for the clinical research,we access its immune effect on pancreatic cancer.Part I Detection of mutation of K-ras oncogene and its significanceObjective : To clone K-ras oncogene,detective its mutate type and site. Methods :There is special expression of K-ras oncogene point mutation in pancreatic cancer, K-ras oncogene was coloned from pancreatic cancer line Bxpc-3, Swl990, Patu8988 by using RT-PCR method.By the means of T-A extension cloning,the gene was ligated with T-Vector and sequenced after purification.Compared with k-ras proto-oncogene in genebank,we find the mutant codon.Results :The production of RT-PCR is about 580bp in agarose gel electrophoresis.There is none of non-specific production.Sequencing results reveal no point mutation in pancreatic cancer line Bxpc-3, Swl990 and 13th, 61th point mutation in Patu8988.We find GGT-GTT in 12thcodon in Patu8988 line and replacement of Gly by Val in K-ras protein. Conclusion :The successful clone of K-ras oncogene and identification of k-ras mutation lays the foundation for the immunotherapy of pancreatic cancer.