Biological Effects Of Lentiviral-mediated Survivin Transfection In Human Degenerative Nucleus Pulposus In Vitro

Objective:To study the biological effects of transforming Survivin gene by lentivirus to human degenerative intervertebral disc nucleus pulposus cells in vitro.Methods:To obtain human nucleus pulposus (NP) cells by cell culture technology. Used Lenti-Sur to transfect the NP cells (experimental group), used blank lentivirus to transfect the negative control group and the blank control group with PBS. The expression of the Survivin gene was analyzed by using Real-Time PCR. The apoptosis rate of the NP cells was detected by flow cytometry. The Caspase-3was detected by microplate reader.Results:The primary NP cells had an average adherence time of7days and took nearly30days to reach90%confluence. The NP cells were polygonal and the cell processes were different. There were2-3nucleolus in the round cell nucleus. MOI=50, the transfection efficiency can reach90%. The growth and the morphology of the cells had no obvious change after transfection, about1week the cell can reach90%fusion; But after passage the growth of the cells became slowly and the morphology of the cells changed, cytoplasm condensed, cell shrinked, cell processes became slender;Real-Time PCR results show that Survivin gene was over-expression in the experimental group(139.55±7.43) compared with the negative control group(1.43±0.16) and the blank control group, and the difference was significant (P<0.05).The apoptosis rate in the experimental group was (2.47±0.13), in the negative control group was (2.38±0.16) and in the blank control group was (2.42±0.26), compared with each other was no significant different (P>0.05).The absorbance of Caspase-3in the experimental group was (0.115±0.01), in the negative control group was (0.101±0.02) and in the blank control group was (0.099±0.01), compared with each other was no significant different (P>0.05).Conclusion:(1) The Survivin gene was successfully transfected into human degenerative NP cells by the lentivirus and over-expression.(2) The growth of NP cells was becoming slowly and the morphological changed, but the cell apoptosis rate was no significant effect.