The Study On Cloning Of Human LIF/NT-3Genes And Its Dual Gene Vector Construction And Expression

BackgroundSpinal cord injury(SCI) is a kind of severe trauma in the central nervous system. It has been known that HUC-MSCs can play the role in the treatment of SCI. Some studies have found that NT-3and LIF gene can repair wounds and maintain neuron survival in the nervous system. So whether the synergistic effect of LIF/NT-3double gene vector and HUC-MSCs can improve the the treatment of HUC-MSCs in diseases, may be a kind of clinical problems to be solved.ObjectivesTo clone human LIF and NT-3gene, and construct the dual expression vector LIF-IRES-NT-3; then integrate the vector into HUC-MSCs genome to make the LIF and the NT-3gene over-expressed in the HUC-MSCs, which may provide a new strategy for stem cell transplantation therapy for the central nervous system diseases, especially for the spinal cord injury.Methods1. Use the overlapping PCR technology and twin PCR technology for cloning the NT-3and LIF gene, which took the human blood mononuclear cell-derived genomic DNA as a template.2. Construsted pIRES2-LIF-EGFP and pIRES2-LIF-IRES-NT-3eukaryotic expressi-on vector through the genetic engineering technology.3. Transfected the screened positive vector into HEK293T cells, to detect expression of the mRNA and protein levels through the RT-PCR and western blot technology.4. Obtained the fragment of LIF-IRES-NT-3and LIF through the genetic engineerin-g thecnology, thus constusted the pCDH-LIF and pCDH-LIF-IRES-NT-3plasmid. 5. Cotransfected the HEK293FT cells with pCDH-LIF/pCDH-LIF-IRES-NT-3combinant plsmid and lentivirus-packaged plasmid pVSVG/Δ8.91, and then collected the supertenant. After enrichment, transfected the lentivirus particles into HEK293FT cells to dectect the expression of LIF and NT-3mRNA and protein levels through the RT-PCR and western blot technology.6. After identification, transfected the HUC-MSCs with packaged lentivirus particles, to dectect the mRNA expression of LIF and NT-3gene.Results1. Cloned the human LIF and NT-3gene successfully. After sequencing, the matchin-g results with Genbank showed the DNA sequence homology of their CDS do main were100%.2. Successfully constructed the pIRES2-LIF-EGFP and pIRES2-NT-3eukaryotic ex-pression vectors, then detected the expressing levels of mRNA and protein incresed clearl-y compared with blank and empty vectors.3. Constructed pCDH-LIF, pCDH-LIF-IRES-NT-3lentivirus expression plasmids suc-cessfully, and obtained the packaged LIF and LIF-IRES-NT-3lentivirus particles, after transfecting the HEK293T with the packaged LIF and LIF/NT-3lentivirus particles, the expresssion of mRNA and protein increased significantly in contrast with the nontransfecti-on group.4. Transfected the HUC-MSCs with the packaged LIF and LIF/NT-3lentivirus partic-les, the expresssion of mRNA and protein also increased significantly in contrast with the non-transfection group.ConclusionsSuccessfully constructed eukaryotic expression vector which contained LIF and LIF-IRES-NT-3gene and also packaged the LIF and LIF/NT-3lentivirus particles. Then transfected the HUC-MSCs with packaged lentivirus vector. The detecting results increased significantly in contrast with the non-transfection group, which indicated that it can overexpress LIF and NT-3.