Study On Anti-Tumor Effect Of MSCs Modified With Gene Transduced By Adenovirus Vector And Leukemia Stem Cell

Objective To construct green fluorescence protein(GFP)labeled recombinant defective adenovirus vector carrying TNFa-Tumstatin fusion gene by using pAdEasy-1 system and transfect it into hUCMSCs, and then observe anti-tumor effect of fusion protein secreted by hUCMSCs modified with TNFa-Tumstatin;leukemia stem cells from mononuclear cells derived from bone marrow of a patient with acute myeloid leukemia(AML)were cultured in vitro and their biological characteristics were investigated in this study.Methods The amplification products of TNFa-Tumstatin by PCR with a pair of primers with Bglâ…¡and Hindâ…¢restriction endonuclease sites and signal peptide sequence of GM-CSF were subcloned into shuttle plasmid pAdTrack-CMV containing GFP gene after digesting with Bglâ…¡and Hindâ…¢.The resultant plasmid,after linearized by digesting with restriction endonuclease Pmeâ… ,was transformed into E.coli.BJ5183 that had been transformed by adenoviral backbone plasmid pAdEasy-1. Recombinant plasmid were obtained by alternation of kanamycin and then confirmed by PCR and restriction endonuclease analysis. Recombinant defective adenovirus vector without objective gene was also constructed as control.The adenovirus vector was packaged and propagated in human embryonal kidney cells(293A cells)after linearized by digesting with restriction endonuclease Pacâ… .293A cells and hUCMSCs infected by the recombinant adenovirus expressed GFP and fusion gene which could be confirmed by fluorescence microscope and RT-PCR respectively,Infection efficiency was analyzed by flow cytometry,fusion protein lever in supernatant of hUCMSCs was detected by ELISA,Suppressive effect to ECV-304 cells and cytotoxicity to U937 cells and THP-1 cells were measured by MTT assay and LDH release assay;The mononuclear cells derived from bone marrow of the leukemic patient were cultured.Morphological characteristics,long-term surviving and proliferative characteristics were observed in vitro.Karyotype and surface markers were investigated by chromosome analysis and flow cytometry.The expression of nucleostemin,Bmi-1,ABCG2,promininl, OCT4 and Nanog in the cells was detected by RT-PCR.Results The results of PCR and restriction endonuclease assay indicated that recombinant adenovirus vector carrying target gene was constructed successfully,Sequence analysis also demonstrated the fusion gene has been insert the vector and Sequence was the same as that of designed fragments.293A cells transfected by recombinant adenovirus expressed GFP and produced virus particles,hUCMSCs could be infected by the virus released from lytic 293A cells and expressed GFP after 24 hours.Along with time porlonged,the fluorescence showed intensive trend,infection efficiency was more than 90%percent in 72 hours. hUCMSCs modified with fusion gene could expressed fusion gene and secreted fusion protein.Inhibition to proliferation of ECV-304 cells and cytotoxicity to U937 cells and THP-1 cells in vitro revealed that the supernatant of these hUCMSCs have distinguished anti-tumor effect.In other study,the cultured cells derived from mononuclear cells of bone marrow of a AML patient grew in a suspended and agminated mode, survived over 12 months and proliferated constantly in vitro. Chromosomal karyotype of the cells was hypotetraploid,nucleostemin, Bmi-1,ABCG2,promininl,OCT4 and Nanog were expressed in the cells. The surface markers on the cells were positive for CD38,CD29,CD44 and HLA-DR,and positive weakly for CD19 and CD71,but negative for CD34,CD13,CD2,CD105.Conclusions The results demonstrate that the replication-deficient adenovirus AdGFP/ TNFa-Tumstatin is successfully constructed by homologous recombination in bacteria and could infected hUCMSCs efficiently.The fusion protein expressed and secreted by hUCMSCs modified with fusion gene displayed expectant anti-tumor function in vitro.The leukemic cells obtained from culture have some characteristics of leukemia stem cells,which laid a foundation for investigation of anti-leukemia effect of hUCMSCs modified with gene at stem cell level.