Study On The Specific Antineoplastic Immunological Effect Of Dendritic Cell Infected By MAGE-A3 Lentivirus Vector In Vitro

Objective: To construct MAGE-A3 lentivirus vector and use it to infect DC which we get it from umbilical cord blood. After that we detect the killing effect of DC to MAGE-A3 positive tumor cell lines and to detect the Anti-tumor effect of DC modified by CTA.Methods:1. Collected the cord blood from full-term healthy puerperal,use gelatin to precipitate red blood cells,and cultured them with cytokines rhIL-4, rhGM-CSF.The phenotype of these DCs was analyzed with morphological assessment structure observing and flow cytometric analysis; The functional properties of these DCs were identified through mixed lymphocyte reaction (MLR) .2. Amplify MAGE-A3 cDNA with gene cloning and RT-PCR technology .And then connect it with lentivirus vector to construct MAGE-A3 lentivirus vector and titer measure.3. Infected imDCs by MAGE-A3 lentivirus vector, induced maturation with rTNF-α.The expression of EGFP in imDC was detected by fluorescent inverted microscope to determin transfection efficency. Detect The gene and the protein expression by RT-PCR and Western-Blot technology. The tumor cell lines which express MAGE-A3 gene were screened out by RT-PCR. Detect the killing rate of CTL against the tumor cells with the expression of MAGE-A3 using the release assay of lactate dehydrogenase(LDH).Results:1. Successfully cultivated more DCs from the cord blood of human being with the united utilization of the cell factors of rhGM-CSF,rhIL-4 and TNF-α.The cells have irregular shape, and possess visible tiny protuberant .The mature DCs show much higher expression of CD83,CD86,HLA-DR than the immature DC. The rate of expression of cell surface molecules including of CD83,CD86,HLA-DR of mature DC were respectively 93.53%, 55.21%, 69.12%. DCs of mature group induced strong allogeneic T lymphocytes proliferation than immature group in MLR (P<0.05) .2. Successfully constructed MAGE-A3 lentivirus vector .Reclaim PCR production with 1% Agarose gel,and we know that the Clips size is 988bp,and the Gene sequencing was the same with MAGE-A3 gene that we needed to construct.Titer measure of MAGE-A3 lentivirus vector was 2×108TU/ml.3. Successfully infected imDCs by MAGE-A3 lentivirus vector. After 12hrs the expression of EGFP was detected,and the transfection rate is ralatived high up to 60%. The gene and the protein expression was detected by RT-PCR and Western-Blot technology. The human gastric cancer cell line(SGC-7901), human hepatocellular carcinoma cell lines(HUH-7) which express MAGE-A3 gene were screen out. Through the release assay of LDH , the killing rates of CTL against SGC-7901 and HUH-7 were 59.98%±4.83%and70.1%±14.4%,which were obviously higher than the corresponding control groups(P<0.05).Conclusion:1. A method to propagate large number of DC from the cord blood in vitro was established.2. We contructed MAGE-A3 lentivirus vector with gene cloning technology.And the drop degree was high.3. The infection rate by MAGE-A3 lentivirus vector was ralatived high. After infection, the DCs could effectively induce MAGE-A3 specific cytotoxic T lymphocytes which could kill MAGE-A3 positive tumor cell lines.4. Our experiment focus on the specific anti-neoplastic immunological effect of dendritic cell loaded MAGE-A3,which has a very important theoretical significance and Value of clinical applications.