Effect Of Mesenchymal Stem Cells Transfected With NPC1 Gene On Proliferation Of Hematopoietic Stem Cells

Hematopoietic stem cells(HSCs)in Niche have the ability of self-renewal and multi differentiation,which can maintain the stability and dynamic balance of hematopoietic system.HSCs are commonly used in clinical transplantation,but they are difficult to obtain,few in quantity and difficult to proliferate in vitro.Therefore,how to effectively promote the expansion of HSCs in vitro is the key problem to be solved.The stromal cells in niche are involved in the regulation of HSCs function through Wnt signal transduction,Notch ligand signal transduction and cytokine secretion.Mesenchymal stem cells(MSCs)are classic niche cells,which can provide essential paracrine factors(growth factors,chemokines,cytokines and exosomes)for the self-renewal and stem maintenance of HSCs in vitro.Lentivirus and other gene modification tools can transfer exogenous genes into MSCs,which can further improve the effect of MSCs on the proliferation of HSCs in vitro.NPC1 is located on the lysosomal membrane and is the key protein for cholesterol transport.Our previous results showed that NPC1gene could promote the proliferation of HSCs.Therefore,in this study,lentiviral particles were used as vectors to transduce exogenous NPC1 into MSCs and stably express it.Through Transwell co culture,the effect of overexpression of NPC1 on the proliferation of HSCs in vitro was studied.The main research contents and conclusions are as follows:(1)Lentivirus preparation:the recombinant plasmid expressing NPC1 gene was constructed.GFP-NPC1 lentivirus and empty lentivirus were prepared by the third generation lentivirus vector system,and purified by ultrafiltration.The infection activity of lentivirus was obtained by flow cytometry,q PCR and titer calculation formula.The results showed that the two lentiviruses were successfully prepared,and both met the concentration range of 10~8TU/m L-10~9TU/m L,with strong transfection ability and could be used for primary cell transfection.(2)Isolation and identification of umbilical cord mesenchymal stem cells:MSCs were isolated from umbilical cord fragments by explant culture method and subcultured to p3.The cells were identified by flow cytometry and differentiation induction.The results showed that Hu MSCs isolated from human umbilical cord grew radially.The high expression of CD90(=100%)and low expression of CD45 and CD34(<2%)were detected by flow cytometry.Osteoblasts and adipocytes were successfully induced.The results met the three minimum characteristics of human mesenchymal stem cells defined by international society for cell and gene therapy.(3)Isolation and identification of hematopoietic stem cells from cord blood:The mononuclear cells from cord blood were separated by density gradient centrifugation,and then the CD34~+cells were separated from the mononuclear cells by immunomagnetic beads,and the purity was identified.(4)Effects of non-contact Transwell co-culture of lentivirus-transfected MSCs and HSCs on the proliferation and morphology of HSCs cells:HSCs group,HSCs+MSCs group,HSCs+GFP-MSCs group,and HSCs+NPC1-MSCs group were divided into three groups.HSCs samples were collected after 5 days of culture.Cell count(×10~5)and Ray-Giamsa staining showed that:HSCs+NPC1-MSCs group(1.51±0.1)were higher than HSCs group(0.44±0.015),HSCs+MSCs group(1.04±0.075)and HSCs+GFP-MSCs group(1.06±0.058),all P values were lower than 0.01.The overexpression of NPC1 in MSCs could significantly improve the proliferation of HSCs in vitro,and there was no significant change in the morphology of HSCs in the four groups.