| AIM: 1) To construct eukaryotic expression vector of human alpha fetoprotein full length gene by RT-PCR method and to verify the new plasmid pEGFP-AFP. 2) To clone AFP cDNA which has Kozak sequence and restriction enzyme digestion sites but no stop code by RT-PCR method. AFP cDNA was ligated with pEGFP-mtP53 vector and named the fusion gene eukaryotic expressing vector pEGFP-AFP-mtP53. The new plasmid pEGFP-AFP-mtP53 was verified and sequenced. 3) To mobilize peripheral blood CD34+ hematopoietic stem cell from patients with hepatocellular carcinoma. And CD34+ cells were induced and cultured into dendritic cells by cocktail cytokines including IL-4,GM-CSFand TNF- a . 4) Transfecting dendritic cells with pEGFP-AFP-mtP53 fusion gene. To evaluate and observe the immune response, which induced by DC after transfected with different genes, against different hepatocellular carcinoma cell lines.METHODS: 1) Hepatocellular carcinoma cell HepG2 was cultured in vitro. Total mRNA was extracted after HepG2 cells were lysated by Trizol. The RT-PCR sense primer was designed including start code ATG, Kozak sequenceand restriction enzyme digestion site Bgl II, while the anti-sense primer was designed including stop code TAA and restriction enzyme digestion site Sal I . Full length AFP cDNA was cloned by RT-PCR method. The vector pEGFP-mtP53/C3 and AFP cDNA were digested by Bgl II and Sal I double digestion. Then AFP cDNA was ligated to vector pEGFP/C3 and the new plasmid was named pEGFP-AFP. The new plasmid pEGFP-AFP was sequenced, and transfected HEK293 cells to be verified. Then cytochemical stain method was use to test the antigenicity of AFP which expressed by transfected HEK293 cells.2) PCR sense primer was designed including start code ATG, Kozak sequence and restriction enzyme digestion site Bgl II, while the anti-sense primer was redesigned with restriction enzyme digestion site Hind III but no stop code. Full length AFP cDNA was cloned by RT-PCR method. The vector pEGFP-mtP53/C3 and AFP cDNA were digested by Bgl II and Hind III double digestion. Then AFP cDNA was ligated to vector pEGFP-mtP53/C3 and the new vector was named pEGFP-AFP-mtP53. The new plasmid pEGFP-AFP-mtP53 was sequenced, and transfected HEK293 cells to be verified. Then cytochemical stain and immuno- fluorescent methods were use to test the antigenicity of AFP and P53 which expressed by transfected HEK293 cells.3) Hepatic cancer patients' peripheral blood CD34~+ hematopoietic stem cells were harvested after mobilized by chemotherapy and G-CSF. CD34~+ hematopoietic stem cell was induced to differentiated into dendritic cell by cytokines IL-4,GM-CSF and TNF- α . The form of DCs were observed and molecules of cell membrane were observed and tested.4) At the fifth day of culture, Plasmid pEGFP-AFP, pEGFP- mtP53, pEGFP-AFP-mtP53 were transfected into dendritic cell using lipofectamine 2000. The immune responses triggered by dendritic cells, against humanhepatic carcinoma cell lines: HepG2, SMMC7721 and MHCC97, were valued through MTT method and the CTL responses were valued between different transfection groups.RESULTS: 1) Eukaryotic expression vector pEGFP-AFP was sequenced and the AFP gene sequence was proved correct. 24 hours After transfected with AFP gene, HEK293 cells expressed green fluorescent protein and cytochemical assay show there were red spots in cytoplasm of HEK293 cells, while the untransfected HEK293 cells were negative. The eukaryotic expression vector pEGFP-AFP was successfully constructed.2) Plasmid pEGFP-AFP-mtP53 was sequenced and the AFP-mtP53 gene sequence was proved correct. 24 hours After transfected with AFP-mtP53 gene, HEK293 cells expressed green fluorescent protein. Cytochemical assay of AFP antigenicity showed that there were red spots in cytoplasm of HEK293 cells. Immuno-fluorescent assay of P53 antigenicity showed that red fluorescence were expressed by transfected HEK293 cells. Double antibody stain immuno-fluorescence assay of AFP and P53 showed that orange, green and red fluorescence were expressed by transfected HEK293 cells. While the untransfected HEK293 cells were negative in all assays. The eukaryotic expression vector pEGFP-AFP-mtP53 was successfully constructed.3) Dendritic cell originated from CD34+ cell had typical dendritic stick and the expression of CD la, CD11C, CD80, CD86 and HLA-DR were increased dramatically after cultured with cocktail cytokines(p<0.05).4) After transfected with AFP-mtP53 gene, dendritic cell expressed green fluorescent protein in 24 hours. Cytochemical assay of AFP antigenicity showed that there were red spots in cytoplasm of DCs. Immuno-fluorescence assay of P53 antigenicity showed that red fluorescence was expressed by transfected DCs. MTT method detected the CTL reaction against HepG2, SMMC7721 and MHCC97 hepatocellular carcinoma cell and showed that transfected dendritic cells can induce AFP and P53 specific immune response. |
