Construction Of Lentivirus Vector Containing Human Homeobox Gene-HOXB4and Its Expression In Human Umbilical Cord Mesenchymal Stem Cells

Objective:To construct lentivirus vector containing human homeobox gene HOXB4and explore changes of human umbilical cord mesenchymal stem cells (HUCMSCs) after infected with HOXB4mediated by lentivirus.Methods:PCR amplification was performed to obtain HOXB4, which was cloned in lenti-shuttle vector. Four-plasmid lentivirus packaging system was used to transfect HEK293T cells. After48hours, lentivirus Lenti-HOXB4was harvested and lentivirus titer was determined. Lenti-HOXB4was used to infect HUCMSCs. The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection (MOI). Meanwhile, RT-PCR, immune fluorescence staining and flow cytometry (FCM) were used to determine the expression of HOXB4and CCK-8was used to determine its effect on cell growth.Results:The results indicated that with Four plasmids were used to co-transfect293T and Lenti-HOXB4was successfully obtained. The determined virus titer was3X108TU/ml; when MOI was20, Lenti-HOXB4had a high transfection rate in HUCMSCs, over80%. In HUCMSCs infected with Lenti-HOXB4, the expression of target gene was detected at mRNA and protein levels. It could promote the HUCMSCs proliferation one times.FCM results showed:CD340.79%, CD451.48%, CD10592.68%, and CD4497.41%. Compared with CD340.13%,CD450.14%, CD10599.95%, and CD9099.88%before transfection, surface markers of hematopoietic cells slightly increased while surface markers of mesenchymal stem cells showed no significant change.Conclusion:1. Lenti-HOXB4and the genetically engineered cell were Successfully Obtained2.The HOXB4gene can promote the high proliferation of HUCMSCs3.HOXB4does not significantly influence the expression of the surface marker of HUCMSCs.