[Expression and biological activity of human neurotrophin-3 in olfactory ensheathing cells mediated by retroviral vector].
We successfully constructed a retrovirus expression vector pRev-TRE-NT3. The supernatant with highest retroviral titers was obtained by transfection of two retrovirus vector, pRev-TRE-NT3 and pRev-Tet-On, into an ecotropic Ecopack-293 cells. Primarily cultured rat olfactory ensheathing cells (OECs) were co-infected with retrovirus following pRev-TRE-NT-3 and pRev-Tet-On system of produced retrovirus. By adding different concentrations of Doxycline, genetically modified OECs were induced to secrete human NT-3. The secreted NT-3 in OEC culture supernatant was detected with western-blot, and its biological activity was confirmed by the growth bioassay of dorsal root ganglion (DRG) cells. The results are as follows: (1) 0.78 kb hNT-3 cDNA was harvested and successfully cloned into pRev-TRE vector by PCR and restriction enzyme digestion methods. The constructed plasmid was identified with electrophoresis, and the direction of hNT-3 cDNA inserting pRev-TRE vector was integrated correctly; (2) NT-3-modified OEC culture supernatant contained 28 kd NT-3 which can bind NT-3 antibody in western-blotting assay, and NT-3 expression was concentration-dependent on Doxycline. NT-3 was not detected in control groups; and (3) Numerous DRG cells migrated from DRG tissue mass in NT-3-modified OECs experimental groups, and these cells had long and thin processes with strong dioptre. These processes formed complicated networks. As for unmodified OEC control groups, a few DRG cells migrated from the tissue mass, and the processes of these cells were rather short. In blank groups, no cells migrated and grew. And their processes in control groups are significantly shorter than NT-3-modified OECs experimental groups. Our results indicate that Tet-On-regulated NT-3 expression in OECs was efficient, offering novel material of in vivo transplantation for the repair of CNS injury.
Yang
Hao
H
Institute of Neurosciences, Fourth Military Medical University, Xi'an 710032.
Jin
Wei Lin
WL
Wang
Chun Ting
CT
You
Si Wei
SW
Ju
Gong
G
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D020933:Neurotrophin 3 / Q000096:biosynthesis* / Q000235:genetics; D009831:Olfactory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012190:Retroviridae / Q000235:genetics
D020933:Neurotrophin 3; D004318:Doxycycline