Experimental Study In Vitro Of RNA Interference On TERT Gene Promotes Spinal Cord Repairment

Objective:To construct recombinant lentivirus vector expressing the small interfering RNA (siRNA) against TERT gene and elavulate its potential for inhibiting the astrocyte. Methods:To construct the recombinant lentivirus, the plasmid pLentilox3.7U6-hTERT was transfected in293T cells. The recombinant lentivirus was verified by PCR. Viruses was purified, propagated in HEK293cells and purified according to the standard techniques,then functional PFU titers were determined. The expression of TERT in astrocyte was detected by qRT-PCR, western blot and immunofluorescence assay. Results:Sequencing test proved that recombinant plasmid was successfully constructed, and through it we successfully packaged lentivirus. The analysis of PCR indicated the recombinant lentivirus containing TERT-siRNA gene. qRT-PCR results showed that:the inhibition rate of TERTmRNA could reach (63.98±2.6)%after Le-TERT infeced astrocytes, and compared with the control group, the difference was statistically significant (P<0.05). Western blot and immunofluorescence assay showed the Le-TERT could effectively inhibit TERT protein expression in vitro. Last but not least, Immunofluorescence staining showed that astrocyte growth was inhibited, with cell apoptosis rate achieves29.4%showed by flow cytometry. Conclusion:Recombinant lentivirus vector expressing siRNA against TERT gene offers a new method and tool for the inhibition of glial scar generation inspired by astrocyte.