Human Bone Marrow Mesenchymal Stem Cells Facilitated The Ex Vivo Expansion And Short-term Engraftment Of Cord Blood CD34+ Cells In NOD/SCID Mice

##无英文摘要内容 ObjectiveTo explore the potential of human bone marrow mesenchymal cells(MSCs) as the feeding-layer to promote ex vivo expansion of cord bloodCD34+ cells and the capacity of expanded cord blood cells to engraftNOD/SCID mice.MethodsHuman bone marrow MSCs were successfully isolated and cultured.MSCs at passage 3 were employed as feeding-layer for the expansion of cordblood CD34+ cell in the presence ofthrombopoietin (TPO), flt3/flk2 ligand(FL), stem cell factor (SCF) and granulocyte-colony stimulating factor(G-CSF). To investigate engraftment potential, unexpanded or expandedcord blood cells were subsequently transplanted into the irradiatedNOD/SCID mice.ResultsAfter 1 or 2 weeks' expansion, there were much more increasing foldsin total nucleated cells (TNC), CD34+ cells and colony forming units (CFUs)in the culture employing MSC as the feeding-layer than those without MSC.In subsequent transplant experiment, we found that the percentage of humanCD45+ cells in bone marrow of the recipient mice transplanted withexpanded cells in the presence of MSC was highest in all the groups sixweeks after transplantation.Conclusion Our study suggested that human MSC enhance expansion of CBCD34+ cells in vitro and their capacity of short-term engraftment inNOD/SCID mice, which may be valuable in future clinical setting. ObjectiveTo obtain high-titer recombinant retrovirus containing HOXB4 gene forthe future research involving hematopoietic stem cells.MethodsRecombinant retrovirus was produced by co-transfection of packagingcell 293T-HOX with plasmids pCMV-G and pCMV-GP. The virus containingmedium (VCM) was subsequently concentrated to increase the titer of retrovirus.And the expression ofHOXB4 gene in the K562 cells infected by the retroviruswas assayed by real-time PCR and western blot.ResultsHigh-titer recombinant retrovirus was obtained by by co-transfection ofpackaging cell 293T-HOX with plasmids pCMV-G and pCMV-GP. And theretroviral titer could be further increased by centrifugation using Eppendorfcentrifuge with moderate virus loss. The target gene could be successfullytransferred into the leukemia cell line K562 via recombinant retrovirus, and theexpression level of HOXB4 in the infected K562 cells was elevated.ConclusionA platform for the retrovirus-mediated gene transfer has been established, which could be applied for the future research conceming hematopoieticstem cells. ObjectiveTo construct recombinant retroviral vector contains both enhanced green fluorescence protein (EGFP) and Hygromycin-resistant (Hyg-R) genes.Methods:Recombinant retroviral vector pCMV-GFP-hyg was constructed by sequentially inserting the fragments of Hyg-R and GFP into the retroviral vector pCMV-LL-SA-2. 293T was used as packaging cells to produce recombinant GFP-hyg retrovirus by co-transfection of 293T cells with pCMV-GFP-hyg, pCMV-G and pCMV-GP.Respite:Recombinant retroviral vector pCMV-GFP-hyg were successfμlly developed. The virus containing medium (VCM) was colleted 24 hours and 48 hours posttransfection, respectively. The titers of recombinant GFP-hyg virus in VCMs were as high as 7.5×10⁵ and 1.5×10⁶ CFU/ml at 24 and 48 hours post-transfection respectively.Conclusions:Recombinant GFP-hyg containing retroviral vector was successfully constructed. ObjectiveTo establish GFP auto-labled leukemia mouse model in new strain ofnonobese diabetic-severe combined immune deficient mice(NOD/SCID-β2m^null .MethodsGFP-expressing K562 (K562-GFP) cells were obtained by infectionof K562 cells with recombinant retrovirus encoded GFP andHygromycin-resistent genes. After total body irradiation with y-ray of 2.5Gy,NOD/SCID mice were intravenously transplanted with 5×10⁵ K562-GFPcells (Group 1, n=3), or 1×10⁶ K562-GFP cells (Group 2, n=3), or normalsaline (Control, n=1). Six weeks after transplantation, the bone marrow,peripheral blood and spleen of recipient mice were assayed for the presenceof human cells by flow cytometry and PCR.ResultsSix weeks posttransplatation, the presence of human leukemia cells was found in the peripheral blood, bone marrow and spleen of recipient mice.The mean percentages of leukemia cells in the bone marrow of recipientmice of Group 1 and Group 2 were 14.5% and 26.6%, respectively.ConclusionHuman leukemia mouse model inoculated with GFP-expressingK562 cells was successfully established, which could be employed in thefuture research.