The Study Of Lentivirus-mediated Swine HOXB4Gene Expressed In Bone Marrow Mesenchymal Stem Cells

Objective Packing swine HOXB4gene recombinant lentiviral and making it infectbone marrow mesenchymal stem cells(BM-MSCs) to discuss the key techniques ofinducing BM-MSCs transform into hematopoietic stem cells(HSCs). MethodsCo-transfected four plasmids into293T cells to prepare swine HOXB4generecombinant lentiviral and made it infect BM-MSCs.(1) Detected the expressionlevel of HOXB4gene using quantitative real-time PCR and western blot after48h the293T cells transfected by swine HOXB4gene expressing plasmid.(2) Co-transfected293T cells for packing swine HOXB4gene recombinant lentiviral using four plasmidssystem and then determine the degree of virus drops.(3) Found out the optimal MOIvalues of swine HOXB4gene recombinant Lentivirus infecting BM-MSCs, then usethe swine HOXB4gene recombinant Lentivirus to infect BM-MSCs at the MOI.72hours later, using quantitative real-time PCR and western blot to detect the expressionlevel of HOXB4gene, quantitative real-time PCR to detect the expression level ofhematopoietic factor RUNX1, GATA2and SCL/TAL1, and flow cytometry to detectthe change of cell cycle phase and observe the shape change of the infected cells.Results (1)48h after the swine HOXB4gene plamid transfected into293T cells, theover-expression of HOXB4gene was detected by quantitative real-time PCR andwestern blot,which indicated that the plasmid was construced successfully;(2)Successfully prepared the swine HOXB4gene recombinant Lentivirus, the virustiter was2×10~9TU/ml;(3) The optimal MOI values of swine HOXB4generecombinant Lentivirus infecting BM-MSCs was30;72h after infection,the HOXB4gene high expressed in BM-MSCs. Significant chang of BM-MSCs was observedunder inverted microscope: cellular pseudopod, which becomes oval or half oval fromlong fusiform gradually, shrunk to cells.72h after the infection, cells in G0/G1phasewere increased significantly, S phase cells were significantly decreased, and the cellswere in the stationary phase; hematopoietic related gene RUNX1and GATA2wassignificantly increased, but SCL/TAL1did not change obviously. Conelusion Ourexperiment successfully prepared swine HOXB4gene recombinant Lentivirus with high titer and found HOXB4gene can induce BM-MSCs shape changes, make thecells in the stationary phase and raise the expression level of hematopoietic relatedfactors GATA2and RUNX1, preliminary discussed the role of HOXB4gene onBM-MSCs, laid a foundation for the subsequent experiment of inducing theBM-MSCs transform into HSCs and effect of HOXB4transfecting into mesenchymalstem cells on differentiation of stem cells on immune related diseases and the HSCstransplantation therapy on tumor and immune related diseases study.