Study Of Cytotoxic Effect Against Esophageal Carcinoma By Dendritic Cells Loaded With HPV16E6/18E7 Gene

Objective: 1. The proliferation of human dendritic cells (DCs) from cord blood CD34+ cells induced by rhGM-CSF, rhTNF-α was observed; meanwhile, the expression of surface molecule CD83 and the activation to T lymphocytes were detected. 2. pcDNA3.1-HPV16E6/18E7 plasmid DNA was abstracted and transfered into DCs for constructing dendritic cells tumor-vaccine. Then the expressions of the aim gene and the activation to T lymphocytes were detected. 3. To detect the type of HPV from esophagea cells EC-109, then, apply the DCs loaded with HPV18E7 gene to kill esophageal cells EC-109 in vitro; to analyse the cytotoxin efficiency of CTL activated by dendritic cells loaded with HPV18E7 gene aganist esophageal cells EC-109.Methods: 1.CD34+ hematopoietic stem cells were isolated from healthy human cord blood using CD34+ progenitor cell isolated kit by a high-gradient magnetic cells sorting system(MACS).The CD34+ cells were expanded with rhGM-CSF and rhTNF-a for 2 weeks. CD83 was analysed by means of flowcytometry and the activation of T lymphocytes by DCs was detected by MTT methods. 2. To abstract the pcDNA3.1-HPV16E6/18E7 by Qiagen Plasmid Mini Kid and use cationic lipid DMRIE-C to transfect DCs. Expression of E6 protein was detected by immuocytochemistry (ICC) and flowcytometry. 3. The typeof HPV from EC-109 cells was checked by dot blot hybridization technique. The two kinds of DCs were cocultured with the T lymphocyte With the presence of relevant cytokines 500ug/ml of IL-2. The activation to T lymphocytes by loaded HPV18E7 gene DCs and the cytotoxic efficiency of CTL aganist esophageal cells EC-109 was detected through MTT methods.Result: 1. About 1.1X1O6CD34+ cells were cultured, expanded and isolated from 50ml cord blood : CD34+ stem cells were cultured in the presence of rhGM-CSF and rhTNF-ot, and the cells were expanded 10~20-fold after 2 weeks. In the culture system, the small colony occur in the 3rd day, when expanded, the colony augment gradually and achieve the largest in the 9th to 10th day. then, with more typical mature DCs occurred, the colony reduced. About 81.7% expanded cells expessed CD83; Human cord blood CD34+ stem cell-derived DCs are capable of stimulating allogeneic T lymphocyte cells to proliferate potentially. 2. The DCs after transfected growth well and develop more dendrite ecphyma. The DCs vaccine was confirmed to express E6 protein by ICC and flowcytometry, and the expression ratio was 49%. Compared with the simple DCs, the DCs tumor vaccine can more effectively stimulate the proliferation of allogeneic lymphocyte in MLR (p<0.01). 3. Transfected with the HPV16E6/18E7 gene, DCs were able to induce potent tumor specific CTL responses. The killing rates of CTLs activated by LAKnsimple DCs and E7-DC were 17.72%, 32.93% and 49.67% respectively. The E7-DCs can kill EC-109 more efficiently than simple DCs (p<0.01). Conclusion: 1. After in vitro culture and amplification, many DCs with typical morphology and phenotype were harvested. Dendritic cells derived from cord blood CD34+ cells are able to differentiate into mature DCs and efficient to induce T lymphocyte in MLR. The cell sorting procedure was simple, reliable. Also, the DCs from human cord blood CD34+ stem cells had very good biological characteristics, especially in immunology. It is the substantial found for DCs applying in application study. 2. Using cationic lipid to transfect HPV16E6 gene into DCs, we successfully generate DCs vaccine, which has efficient biological activity in vitro. It is an asepsis and efficient transfection method in adopting cationic lipid DMRIE-C to transfect DCs. So, we do some beneficial explore in basing on DCs for anti-tumor immunity. 3. Transfected with the HPV18E7 gene, DCs were able to induce potent tumor specific CTL responsiveness more than simple DC. Our researches demonstrate that DCs-vaccine loaded with HPV18E7 gene is a promising approach for esophageal cancer gene therapy based on DCs. The results are very exhilarating, so it may become a new model and optional model for tumor gene/ DCs therapy clinical application in anti-esophageal cancer.