Gene Therapy Of Hepatocellular Carcinoma Via Endothelial Progenitor Cells Armed With A Suicide Gene: A Pilot Study In Nude Mice

Part-1 Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133+Enriched CellsObjective:Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133+enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs.Methods:PBMCs from healthy subjects were used either directly for cell culture or for CD133+sorting. The two groups of cells were cultured in complete medium M199 for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo.Results:The results showed that PBMCs produced more colony-forming units (CFU) than CD133+enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+cells in CD133+group were less than in PBMCs group (P＜0.01). PBMCs group secreted more VEGF than CD133+group on the day 7 (P＜0.01). As compared with CD133+group, PBMCs group had more potent potential of proliferation and vascularization in vitro.Conclusion:It was concluded that CD133+sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, speculating that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.Part-2 Influence of Cobalt Chloride Preconditioning on The Proagiogenic Function of Endothelial Progenitor CellsObjective:To exploit the effect of cobalt chloride (CoCl2) pretreatment on angiogenic function in vitro of cultured endothelial progenitor cells(EPC).Methods:Cultured endothelial progenitor cells from patients were pretreated with CoCl2 under a range of concentrations. Then, Cell proliferation and VEGF secretion were examined by MTT and ELISA. Matrigel experiment and migration assay were imitate vascularization in vivo.the expressions of HIF-la and its target genes were examed by Western blot analysis.Results:CoCl2 pretreatment with 100μM at 6 hours enhanced the angiogenic function of EPC such as secretion, chemotaxis and tube formation in vitro, CoCl2 pretreatment with more than 400μM or 24h hinderd the functional properties of EPC, the ratio of BCL-2/BAX decreased along with an increasing concentration of CoCl2.Conclusions:CoCl2 preconditioning with low concentration and short time will enhance neovascularization of EPC in vitro, high concentration of CoCl2 reduce cell viability and proliferation, which may attributable to increased expression of proapoptotic gene. Part-3 Pilot Research on Targeted Therapy for Transplanted Human Hepatocarcinoma under Nude Mice Skin Based on Endothelial Progenitor Cells Armed with A Suicide GeneObjective:To explore the killing effect of gene modified EPC (EPC-CD/UPRT) on hepatocarcinoma HepG2 and SMMC-7721, established a transplanted tumor model of human hepatocarcinoma in nude mouse and study the killing effects of direct EPC-CD/UPRT treatment and pretreatment with cobalt chloride.Methods:pORF5-Fcy::Fur plasmid with its correctness evaluated by the means of restriction enzyme and destination protein detected by immunohistochemistry after transduction to EPC with fugene HD, HepG2/SMMC-7721 cells were mixed with modified EPC by different proportion and the total cell survival rate was analyzed with MTT. Hepatocarcinoma cells then be inoculated into the neck skin of nude mice. When the tumor is formed, the damage effects of EPC-CD/UPRT under the 5-fluorocytosine can be observed. The depressor effects in tumor with direct injection of EPC-CD/UPRT or pretreatment with cobalt chloride before application was compared, and then the tumor sizes and tumor inhibitory rate was calculated; finally, tumor Apoptosis Index was tested with TUNEL assay.Results:Enzymes electrophorogram shows that plasmids are extensively increased successfully with the purity of 99%; IHC results shows that after gene transformation, EPC can express the target protein CD; MTT analysis indicate that EPC-CD/UPRT can play a significant role of bystander effects which cause obvious damage to HepG2 and SMMC-7721; EPC-CD/UPRT mixed with 5-FC can effectively restrain the growth of transplanted tumor of nude mice; the pretreatment of cobalt chloride can enhance the inhibitory effects of tumor; CD/UPRT/5-FC system may play a proapoptosis role in tumors.Conclusions:EPC armed with CD/UPRT can effectively inhibit the growth of hepatocarcinoma transplanted tumor. The pretreatment of cobalt chloride may enhance the restraining effects by recruitment more EPC-CD/UPRTs...