| Objective:1.to establish the monoclonal antibodys against human FXYD3 and identify its biological characterization.2.to investigate histological distribution of human FXYD3 in tumor of hepatobiliary system and to explore the correlation between expression level and biological proceeding so as to reveal potential effective mechanism during the course of tumor cell progression.3.to isolate ricin toxin from seeds of Ricinus Communuis and to purify, identify its biochemistry characterization.4.to compare cytotoxcity effect of ricin on different hepatobiliary tumor cell hnes and to study the effects of ricin on FXYD3 expressive level in tumor cell lines to get the experimental evidence for succedent study on immunotoxin of monoclonal antibody to human FXYD3 and ricin.Method:1.dominant epitopes of FXYD3 were analyzed by software BioSunV1.0,two peptides were synthesized and conjugated with KLH.BALB/c mice immunized with KLH-conjugated peptide.The hybridoma cell lines were selected in CM semisolid culture medium containing HAT.The selected hybridoma cell and its monoclonal antibody were identified by ELISA,SDS-PAGE,Western Blot and ICC.2.Using the McAb to detect the FXYD3 expression in tumor cells of HepG2,Hcccc-9810,BxPc-3 and tumor tissues by the method of ICC,IHC and Western Blot to draw the qualitation conclusion,Real-time PCR to draw the quantitation one.3.Ricin was isolated using the method of Nicolson and Blaustein and purified using Sepharose 4B affinity chromatography.Ricin was identified by SDS-PAGE electrophoresis.4.MTT was used to detect and compare the cytotoxicity of the Ricin to different cancer cell lines HepG2,Hcccc-9810 and BxPc-3.FCM was used to analyse cell cycle and the cell death caused by ricin.Cells were cultured with ricin in 24 hours to study the effects of ricin on FXYD3 expressive level in these cells.Result:1.four hybridoma cell lines producing high titer human FXYD3 monoclonal antibodys were obtained.All antibodys were identified to be IgG1 subtype and showed high affinity and high specificity to human FXYD3.One of McAb 76-02F1E8 got the titer with 1:105 and its affinity constant was 3.11×108L/mol.2.FXYD3 expressed in tumor tissues of HCC,cholangiocarcinoma,pancreatic carcinoma,gastric carcinoma,mammary adenocarcinoma.FXYD3 expressed differentially in HCC,cholangiocarcinoma,compared with circa- carcinoma tissues respectively and the protein expression level is correlated to tumor cells' differentiated degree in cholangiocarcinoma.FXYD3 expressed degressively progressively in HepG2,Hcccc-9810 and BxPc-3.3.10mg Ricin with the concentration of 1.3mg/ml was isolated from seeds of Ricinus Communuis.4.The ricin has the similar cytotoxicity to HepG2,Hcccc-9810 and BxPc-3,the IC50 of ricin to these cells is 0.637×10-9mol/L,0.366×10-9mol/L and 0.535×10-9mol/L respectively.Ricin can induce Hcccc-9810 to stay in G0/G1 phase in 24 hours.Different concentrations ricin has cause Hcccc-9810 and BxPc-3 to express FXYD3 less than blank group in different degree.Conclusion:1.FXYD3 maybe associated with carcinogenesis and progression of HCC, cholangiocarcinoma,pancreatic carcinoma,gastric carcinoma and mammary adenocarcinoma. 2.FXYD3 may be one important clinical subsidiary marker on cholangiocarcinoma prognosis. 3.The measure that FXYD3 was detected combined with AFP and CA19-9 respectively may be helpful to increase diagnosis rate of HCC and cholangiocarcinoma respectively.4.The different sensitivity of HepG2,Hcccc-9810 and BxPc-3 to Ricin's cytotoxicity is no statistical significance.5.Ricin can lead Hcccc-9810 and BxPc-3 cells to express FXYD3 less.
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