Targeting Therapy Of Marrow-derived Endothelial Progenitor Cells With CD Transfceted On Lung Cancer

Lung cancer is a disease with high morbidity and mortality. Themajority of patients with lung cancer are diagnosed at advanced stage.The suicide gene therapy is one of the most promising gene therapies forthe tumor， due to its directly tumor killing effect. The objects of ourstudies were to investigate the killing action on the co-cultured lungcancer cell line SPC-A1in the occurrence of autophagocytosis and therelated bystander effect of gene modified type endothelial progenitor cells(EPCs) group induced by prodrug5-fluorocytosine (5-FC). In our paststudy， EPCs in the bone marrow could be directed to the tumor andparticipated in the establishment of tumor angiogenesis. Then， therecombinant adenovirus expressed CD-DNA-TPC was successfullytransfected into EPCs and gene modified type EPCs were obtained.Under the different concentrations of5-FC， cell growth inhibitory rateof CD gene in modified type EPCs was lower than that in wild type EPCs.By the co-culture of gene modified type EPCs and lung cancer cell lineSPC-A1， CD gene was specifically delivered to the lung cancer.Administration of5-FC not only induced autophagocytosis but also triggered bystander effect， which resulting in a significantly anti-tomoreffect. Our data showed that combination of EPCs and suicide genetherapy can effectively improved the damage efficiency of suicide gene.Our experimental results provided theoretical and experimental basis forexploring new lung cancer treatments. Objective: To analyse the segregation and cultivation methods of marrow-derived EPCs for futher research.Methods: Mononuclear cells were segregated from the marrow of micethrough density gradient centrifugation and then cultivated on humanfibronectin coated plates.After that， these cells growed under thestimulus of vacular endothelial growth factors and recombinant humanepithelial growth factors were purified the EPCs.The EPCs can uptakeFITC-UEA-I and Dil-ac-LDL. The expression of EPCs surface markerswas detected by means of flow cytometry.Results: EPCs were successfully separated from the marrow of mice. Thegrowth of EPCs beared the characteristic of colony formation and pavingstones.EPCs could intake FITC-UEA-I and Dil-ac-LDL and there was noremarkable difference in the double positive chromosomal cells. A highexpression of VEGF-2and CD133in EPCs was displayed.Conclusion: It is a workable method with obvious advantages to separatefrom mice marrow EPCs， thus providing a theoretical basis for furtherresearch and future experiments. Objective: To investigate the killing action on the co-cultured lungcancer cell line SPC-A1in the occurrence of autophagocytosis and therelated bystander effect of gene modified type endothelial progenitor cells(EPCs) group induced by prodrug5-fluorocytosine (5-FC).Methods: In vitro cultured human bone marrow EPCs were separated byusing gradient centrifugation， cytosine deaminase (CD) gene modifiedtype EPCs were obtained. The expression of CD gene in gene modifiedtype EPCs was detected by RT-PCR. The cell survival rate of genemodified and wild type EPCs in different5-FC concentration wereobserved and compared， the bystander effect induced by gene modifiedtype EPCs was also investigated.Results: Eucaryotic cells expressed CD-DNA-TPC was successfullytransfected into EPCs and CD gene was positive in gene modified typeEPCs. Under the different concentrations of5-FC (0.01~1000μg/ml)，the cell growth inhibitory rate in different ratio of co-cultured CD genemodified type EPCs was lower than that in wild type EPCs. Thedifference between2groups was significant under the same concentrationof5-FC exposure (p<0.05，for all samples). After72h culture， the cellgrowth inhibitory rate in different ratio of co-cultured CD gene modified type EPCs and human lung cancer cell line was higher than that of genemodified type EPCs. When the ratio reached50%， the cell growthinhibitory rate was already more than70%.Conclusion: After human bone marrow EPCs transfected with CD gene，the administration of5-FC can induce autophagocytosis and triggerbystander effect， as well as kill the co-cultured human lung cancer cellline SPC-A1. Our data will shed lights on the further work for therapeuticmodalities of combined anti-angiogenesis agent with suicide gene therapyin vivo. Objective: To discover the apoptosis and the inhibiting function of EPCscontaining common CD towards the growth of subcutaneous transplantedtumors of nude mice; to explore the feasibility of exploiting CD-EPCs forthe targeted therapy of lung cancerMethods: Four groups of3-5week BALB/C rates were randomlydivided:every group has30(n=30).Every groups were injected SPCA-1cells in subcutaneous.Three days later，different groups were injecteddifferent cells transplantion into the nude mice via tail veins:group A forCD-EPCs; group B for EPCs; group C for CD adenovirus;group D justPBS.From than，the7th day to14th day5-FC（10mg/500μL） wereinjected into all the nude mouse via tail veins once a day and the weightand volume were recorded.Besides，Tunel test was exploited to detect thepoptosis rate of cells in tumour tissue.Record the death time of remainingrates of every group and draw the Kaplan-Meier graph.Results: The weight and volume of rate tumor in the C group which wasEPCs transfered with CDgene (CD-EPCs)apparently lower than the othergroups (p<0.05). Detected by means of Tunel， the apoptosis rate of cellsin the CD-EPCs group was25.28±3.12%， evidently higher than theother groups (p<0.05). Suvial time has no apparently different. Conclusion: EPCs can be used as the carrier for the targeted therapy oflung cancer and display desirable tumor-inhibiting effects through armedsuicide gene(CD)， thus providing prospects for the treatment of lungcancer.