Experimental Study On Construction Of Lentiviral Vector Carrying The TNF-α Gene And Its Expression In The UCBMSCs

AIM:To construct a lentiviral vector carrying human TNF-a gene, and observe its expression in the human umbilical cord blood mesenchymal stem cells.Methods:the human TNF-a gene was obtained with reverse transcription polymerase chain reaction (RT-PCR) from the PCD DNA-TNF-a plasmid, the TNF-a gene was recombined to construct the transfer plasmid pGC-FU-TNF-a by infusion technique, the transfer plasmid pGC-FU-TNF-a, the construction plasmid Helper1.0 and the envelope plasmid Helper2.0 were contransfected to 293T cells mediated by lipofectamine 2000 to produce lentiviral particles. the human umbilical cord blood mesenchymal stem cells were divided into the pGC-FU-TNF-a group (experimental group, EG),the pGC-FU-EGFP group (mock group, MG) and the ucbmscs group (blank group, BG) were Separately infected by obtained lentiviral particles, Lentiviral particles and PBS,the expression of TNF-a was observed by RT-PCR and Elisa.Result: The result of sequencing showed that the cloned TNF-a gene was consistent with the sequence reported in Gene Bank. The insertion of TNF-a gene in viral genome was conformed by PCR. The pGC-FU-TNF-a plasmid was identified to have correct sequence. After the three plasmids of lentiviral vectors were contransfected to the 293T cells. The supernatant was collected and concentrated. The titer of the lentiviral vector particles was found to be 1×107TU/L. After the constructed lentiviral vectors infected the ucbmscs, the results showed that there were the expression of TNF-a mRNA in the three groups by the RT-PCR, the pGC-FU-TNF-a group (2.71±1.57),the pGC-FU-EGFP group (4.63±1.32) and the ucbmscs group (6.03±0.88) have significant difference between the three group (P<0.001),the expression of TNF-a by Elisa show that the pGC-FU-TNF-a groups (1.32±0.23 ng/ml) the pGC-FU-EGFP group (0.70±0.25ng/ml) and the ucbmscs group (0.76±0.11 ng/ml) have significant difference between the three groups (P＜0.001).Conclusion:(1) Lentiviral vector carrying TNF-a gene has been constructed, product hight recombined lentiviral virus. (2) Lentiviral virus infected human umbilical cord blood mesenchymal stem cells and the TNF-a protein could be expressed. (3)This will facilitate the following exploratory development of TNF-a gene modified stem cell-based gastric carcinoma.