The Observation And The Study On Differential Proteins For Interaction Between Nasopharyngeal Carcinoma Cell And Lymphatic Endothelial Cell

Objective:To observe the interaction of highly metastatic cell line of nasopharyngeal carcinoma(5-8F)and lymphatic endothelial cell(LEC)in vitro after the co-cultured and to search differentially expressed proteins of LEC and 5-8F cells after co-cultured.To lay the foundation for the study of the molecular mechanisms of lymph node metastasis in nasopharyngeal carcinoma.Method:First,GFP-LEC and RFP-5-8F cells which were transfected with lentivirus marked fluorescent protein were co-cultured in vitro by three methods.The change of movement,migration and morphology of cells was observed through live cell station.Then,the conditioned medium(CM)of LEC and 5-8F cells after cultured 24 hours were prepared,respectively.MTT array was used to value the effects of 5-8F-CM with different concentrations on LEC proliferation and wound healing test was used to evaluate the migration of 5-8F after using LEC-CM with different concentrations.Flow cytometry was used to separated green fluorescent protein-LEC(GFP-LEC)and red fluorescent protein-LEC(RFP-5-8F)after co-cultured directly.We also detected differentially expressed proteins of GFP-LEC and RFP-5-8F before and after co-cultured by using protein chip.According to the detected results of protein chip,we selected Fractalkine and insulin-like growth factor II receptor(IGF-IIR)to verify the accuracy of protein chip by immunohistochemistry(IHC)technology.At last,we analyzed the differentially expressed proteins by using bioinformatics technology.Result:1.Through the observation under live cell station,we found that during co-cultured,some RFP-5-8F cells stretched pseudopodium,became fusiform and got higher ability of movement and migration.Some RFP-5-8F cells also had bigger size and lamellipodia.Moreover,we also observed some RFP-5-8F cells grew by separated each other manner instead of getting together.GFP-LEC trending to surround RFP-5-8F cells became tubular and had more obvious proliferation.At last,suspected fusion phenomenon between RFP-5-8F cells and GFP-LEC was observed.2.The result of MTT showed that no statistical difference of LEC proliferation was found when comparing all 5-8F CM concentration groups with their corresponding with control group(P>0.05)after co-cultured 24 hours,respectively.After co-cultured 48 hours,the proliferation ability of LEC was promote by the groups of 5-8F CM with 10%,50%,75%and 98%concentration levels(P<0.05).After co-cultured 72 hours,only 75%and 98%concentration 5-8F CM promoted LEC proliferation(P<0.05).The result of scratch wound healing indicated that the migratory distance of 5-8F cells dealt with LEC CM of each concentration was greater than the control group after co-cultured 6,12 and 24 hours(P<0.01).3.The result of protein chip showed that there were 328 differentially expressed proteins of GFP-LEC and 177 differentially expressed proteins of RFP-5-8F after co-cultured.The analytical result to IHC suggested comparing with control group,the expression of Fractalkine and IGF-IIR was increased and the difference was statistically significance(P<0.05).4.The analytical result of bioinformatics to differentially expressed proteins of RFP-5-8F and GFP-LEC showed these differentially expressed proteins mainly involved in proliferation,adhesion and migration process.Conclusion:1.After co-cultured,there was interaction between LEC and 5-8F cells,which promoted the proliferation of LEC and the migration of 5-8F cells.2.The interaction between GFP-LEC and RFP-5-8F associated with tumor metastasis process.