The Expression Of Chemokines CCL19,CCL22 And Cytoskeletal Proteins Fascin-1 By Dendritic Cells Pulsed With K-ras Mutant Peptide

Objective:Malignant tumor of human beings occurrence and development may be one of the most complex disease process, many cellular and molecular mechanisms have been explained:Dendritic cells (DC) dysfunction has been proven to be one of the important reasons causing cancer immune deficiency/escape. DC are the most powerful antigen presenting cells, they exist in a variety pf organs and tissues. Dendritic cells need to migrate while completing the functions. They activate the initial T cells when reaching the lymph nodes, cause immune response and kill the tumor cells and inflammatory molecules. It is precisely because DC has such a powerful feature, that DC has been modified into a variety of tumor vaccines to enhance the ability to eliminate tumor. Therefore, the research of a variety of DC cancer vaccine has become a hot spotand now has completed a number of randomized clinical trials, but its efficacy is not reported often in conjunction with the performance of side effects such as fever, and need to be further explored and improved. Molecular such as chemokines always played a regulating> promoting or inhibiting role in the processing of completing the functions and migration. Their relations are very complicated.To explore the expression of cytokines CCL19,CCL22 and cytoskeletal proteins Fascin-1 on the K-ras mutant peptide pulsed dendritic cells, for clinical application of K-ras mutations in tumor peptide vaccine of dendritic cells providing a theoretical basis.Methods:Peripheral blood of healthy volunteers collected sterile 25ml, with 2.5ml heparin, mononuclear cells were isolated with lymphocyte separation medium, and then washed two times with RPMI1640. Put the cells into the containing which the volume fraction of 10% of the AB-type serum RPMI1640 in the diluted 2×106/ml,then add 6-well plate,37℃,5% CO2 cultured for 2 hours. Adherent cells were washed three times with RPMI1640(pH7.0), washing away the non-adherent cells. Added the culture medium containing RPMI1640,10% fetal bovine serum, GM-CSF1000u/ml, IL-4 500u/ml into the culture plate, changed a half the amount of the culture medium in the third day. The seventh day, cells were collected (1x106/ml), and devided two groups:A group:No loading of dendritic cells derived from peripheral blood, B group:K-ras mutation in peptides (50μg/ml) loading dendritic cells derived from peripheral blood, adding TNF-a (lOng/ml) to two groups respectively,continuing to induce 2 days. Phenotype(CDla, CD80, CD86) were identified by flow cytometry,. Respectively, the morphological structure were observed through the inverted microscope,scanning electron microscopy and transmission electron microscopy. The expression(absorbance) of IL-12, CCL19 and CCL22 were examined by Enzyme-Linked Immunosorbent Assay(ELISA) continuously(before loading,6h,12h,24h,48h). The expression of cytoskeletal proteins were determined by Western blot.Results:The expression of CD1a,CD80,CD86 were identified highly in the loading group after K-ras mutations peptide loading DC, before and after loading the average expression rate of CD1a, CD80 and CD86 are as follows:38.1%,64.3%,61.4% and 70.2%,90.5%,91.6%, there is statistically significant (P<0.01). Great changes were happened in the morphological structure:No loading group DC:dendritic processes relatively short and sparse; loading group DC:cell processes thickness range of different lengths, patterns vary widely, and some showed branch shape, and some showed petal-like. Nuclei clear and well-developed endoplasmic reticulum were observed under transmission electron microscope. The expression of IL-12,CCL19 and CCL22 in the two groups were almostly the same before K-ras mutations peptide loading DC (P>0.05).The expression of IL-12, CCL19 and CCL22 in the loading group were higher almostly than that in the non-loading group (P<0.01) As the time going, the expression level did not increased obviously in the non-loading group, while the expression level were continuing increased significantly in the loading group, and showed an upward trend. Western blot results:the expression of cytoskeletal proteins secretion by the Lab-wiok4.6 professional software analysis of gray between the two groups with significant statistical significance (P<0.01).Conclusions:K-ras mutant peptide can promote DC to mature, improve the expression of cytokines and cytoskeletal proteins and strengthen DC migration, thereby enhancing the anti-bacterial or tumor cells. K-ras mutations in peptide load-DC can be used as a dendritic cell tumor vaccines, in order to lay the foundation for future research and thus for clinical use.