Generation Of Human Dendritic Cells From Cord Blood CD34+Cells In Vitro And The Recombination Of PcDNA3.1-HPV18E7 (pcDNA3.1/myc-His<一>A-HPV16E6)

Objective : (1) To observe the proliferation and biological characteristics of human dendritic cells from cord blood CD34+ cells in presence of rhGM-CSF, rhTNF-. (2)To construct pcDNA3. 1-HPV18E7 (pcDNA3.1/myc-His<->A-HPV16E6 ) with HPV18E7(HPV16E6) and pcDNA3. 1 (pcDNA3.1/myc-His<->A). 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, rhTNF- for 2 weeks. Many techniques including ordinary light microscopy, FACS and MLR were adopted in our experiments to detect the biological characteristics of DCs. (2)To abstract the HPV18 DNA(HPV16 DNA) from the infected cells by HPV18 (HPV16) , amplify the HPV18E7 DNA(HPV16 DNA)by PCR, construct the pcDNA3. 1-HPV18E7( pcDNA3.1/myc-His<->A-HPV16E6 ) with HPV18E7 ( HPV16 ) and pcDNA3. 1(pcDNA3.1/myc-His<->A) by gene recombination technique. Results : (1) Percentages of CD34+cells purified from cord blood MNC were 0. 5%~0. 9%. (2) Human cord blood CD34+ cells were cultured in the presence of rhGM-CSF and rhTNF-a, and the cells were expanded 6~ 10-fold after 2 weeks. The purity of DCs is more than 90%. (3)The culture condition led to the occurrence of small adherent aggregates in the third day. when expanded, aggregates relased DCs with typical morphology in the medium during the 5th - 7th day. After the 7th ~ 10th day aggregates reduced , with more typical morphology DC. (4)About69% expanded cells expressed CD83. (5)Human cord blood CD34+ stem cell-drived DCs potentially stimulated allogeneic T cells to proliferate. (6) The result of PCR and sequencing proved that the product was HPV18E7( HPV16E6),and PCR, sequencing test also proved that the recombinat was pcDNA3. 1-HPV18E7 (pcDNA3.1/myc-His<->A-HPV16E6. Conclusions: (1) A lot of DCs with high purity and typical morphology could be generated and activated to be mature by culturing human cord blood CD34+ stem cells with rhGM-CSF, rhTNF- in vitro. 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. (2)The recombination of pcDNA3. 1-HPV18E7 ( pcDNA3.1/myc-His<->A-HPV16E6 ) would benefit to the treatment of tumor related with HPV, such as esophageal carcinoma.