In Vitro Study On CD-UPRT Gene Therapy For Malignant Glioma Using Endothelial Progenitor Cell Vehicle Derived From Human Peripheral Blood

Malignant gliomas are one of the most common brain tumors.They are also among the most angiogenic solid tumors in human being.Because of their highly invasion and recurrence,local treatments,such as surgical resection and stereotactic radiosurgery,only have limited impact on survival, whereas systemic therapy(e.g.chemotherapy and immunotherapy)have been rather disappointing.The poor prognosis of patients with malignant brain tumors has led to the search for novel therapeutic strategies in spite of aggressive multimodality therapy.Recently,as more and more the understanding about tumor genesis and development,we have learned that cancer development is a complex event involved multifactor,multigene and multiphase.Respectively,new therapeutic theories and methods should be developed to conquer cancers by multiapproach,multitarget and multiphase.We supposed that EPCs could be acquired through expanded mononuclearcells which were separated from peripheral blood by density gradient centrifugation,and cultured EPCs could be transfected as well with eukaryotic expression vector bearing combined suicide gene CD-UPRT. EPCs could home to the tumor,integrate themselves into vascular endothelia and incorporate into surrounding tumor tissue.The pro-drug 5-FC was given to induce EPCs apoptosis resulting in the inhibition of tumor angiogenesis,meanwhile the apoptosis could trigger bystander effect by which glioma cells could be killed.Under above background,we designed this research to investigate the feasibility of combined anti-angiogenic therapy with suicide gene therapy for malignant glioma.In this research,we intended to acquire EPCs from peripheral blood and expand them in vitro,then trasfected them with plasmid pCEA CD CAUP bearing CD and UPRT gene,at last evaluated CD-UPRT expression in modified EPCs population as well as the effect of CD-UPRT/5-FC induced cell death.In our research,we cultured mononuclearcells obtained from human peripheral blood by density gradient centrifugation in EGM-2.On 7th day most of cells began to acquire spindle-shaped,endothelial cell-like morphology and endothelial characteristics such as ac-LDL taking up and UEA-1binding.CD34,CD133,and VEGFR-2 expression were detected by immunofluorescence in cultured EPCs.By flow cytometric analysis,the results of quantitation of different immunopositive cells in cultured EPCs were as follows:37.645%of CD34~+,45.655%of CD133~+,57.3%of VEGFR-2~+,29.44%of CD34~+ and CD133~+ double positive,28.38%of CD34~+ and VEGFR-2~+ double positive.The number of EPCs on day7 was augmented 100 times than those on day1.We demonstrated the adhesion and migration abilities of EPCs by adhesion and Transwell experiments.We introduced plasmid pCEA CD CAUP bearing suicide gene CD-UPRT into EPCs by lipofectamin2000 and detected CD and UPRT expression by RT-PCR successfully.We compared survival rate of gene modified EPCs with unmodified EPCs' in serial diluted 5-FC respectively to prove that gene modified EPCs population expressed CD and UPRT gene exactly.The result showed that there was no significant toxic effect on unmodified EPCs cultured in the serial dilution of 5-FC from 0.001 to 1 000μg/ml,but the survival rate of gene modified EPCs was below 50%in 10μg/ml of 5-FC at 72h.If the concentration of 5-FC increased to 100μg/ml,modified EPCs survived hardly.Based upon this result,100μg/ml of 5-FC was chosen in bystander effect assay in this research.To observe bystander effect,we mixed modified-EPCs population and U251 cells with different ratio,exposed to 100μg/ml of 5-FC for 72h. Bystander effects were found in every group and augmented following the increase of modified-EPCs proportion mixed with.The cell growth inhibition rate was over 80%when half of cells were modified EPCs. In short,in this research,we separated,expanded and identified EPCs from human peripheral blood.After transfected it with plasmid pCEA CD CAUP by lipofectamin2000,we detected that modified EPCs population expressed CD and UPRT gene exactly,and observed the toxic effect of pro-drug 5-FC on modified EPCs population.In bystander effect assay,we demonstrated bystander effect was augmented while percentage of modified EPCs population was increasing.All of results in this research would support further work for malignant glioma therapy with suicide gene using human endothelial progenitor cell as a vehicle.