Anti-melanoma Effect Of Survivin-specific Lymphocytes Combined With CCL17 And GM-CSF

Malignant melanoma is the most deadly skin cancer,and the clinical treatment of melanoma is not satisfactory.There are many methods used to treat melanoma,and tumor immunotherapy has received much attention as a new generation of therapies.Among them,specific immunotherapy against tumor antigens has been proven to have a good effect in the treatment of melanoma.Survivin antigen is a tumor-associated antigen and is a potential therapeutic target.Inducing its specific cytotoxic lymphocyte（CTL）may have a certain therapeutic effect on melanoma.In addition,two cytokines,GM-CSF and CCL17,may play an important role in the function of CTL.Therefore,this thesis studies the anti-tumor effect of Survivin-specific CTL combined with GM-CSF and CCL17,aiming to provide some reference and help for clinical immunotherapy of melanoma.Part Ⅰ Bioinformatics analysis and in vitro cytology experiments of Survivin epitopesOBJECTIVE:To analyze the mouse Survivin gene by bioinformatics on three websites,and then to culture Survivin-specific lymphocytes.Method:UsingthreeonlinesitesBIMAS（https://www-bimas.cit.nih.gov/molbio/hla_bind/）,SYFPEITHI(http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htmAnd IEDB 3.0（http://www.iedb.org/）to score and predict the mouse Survivin（Birc5）gene and the 9-amino acid epitope peptide encoding the entire amino acid sequence,and select the peptide with higher score for synthesis.The peptide vaccine was used to immunize normal mice,then the spleen lymphocytes were isolated,and Survivin-specific CTL was induced to act on B16F10 cells,and GM-CSF and CCL17 proteins were added to the culture system.The effects of the combination of the three on B16F10 cells were examined by Calcein-AM method and flow cytometry.RESULTS:Two9-peptides of Survivin_66-74（GWEPDDNPI）and Survivin_85-93（AFLTVKKQM）were screened,and Survivin-specific CTL was successfully induced.Moreover,the addition of CCL17 protein increased the specific cleavage rate of CTL to B16F10,while the addition of GM-CSF protein did not increase significantly.Conclusion:In vitro combined with Survivin-specific CTL and CCL17 have stronger killing effect on B16F10cells,while combined with GM-CSF does not enhance the killing effect of CTL on B16F10 cells.Part Ⅱ Construction of a B16F10 cell line stably expressing CCL17 and GM-CSFOBJECTIVE:To construct a B16F10 cell line stably expressing CCL17and GM-CSF for preparation in subsequent experiments.Methods:Primers of GM-CSF and CCL17 were designed,and then GM-CSF and CCL17genes were introduced into B16F10 cells by a series of genetic engineering methods and expressed.RESULTS:By enzyme digestion,sequencing and the like,we demonstrated that GM-CSF and CCL17 were successfully introduced into the expression vector and successfully expressed in B16F10cells.Conclusion:The B16F10 cell line expressing CCL17 and GM-CSF was successfully constructed,which laid a foundation for subsequent experiments.Part Ⅲ Effect of Survivin-specific CTL combined with CCL17 and GM-CSF on mouse melanomaOBJECTIVE:To investigate the effects of adoptive infusion of Survivin-specific CTL combined with CCL17 and GM-CSF on melanoma in mice.Methods:Mouse melanoma was used as a model to observe the effect of the combination of the three on tumor volume and tumor weight.The infiltration of local lymphocytes was detected by immunostaining and immunohistochemistry.RESULTS:Survivin-specific CTL combined with CCL17 and GM-CSF could better inhibit tumor growth.HE staining and immunohistochemistry showed that the tumor had the most local necrosis and more infiltrating lymphocytes.Conclusion:In vivo experiments show that Survivin-specific CTL combined with GM-CSF and CCL17 can inhibit the growth of mouse melanoma.