EXPRESSION AND BIOLOGICAL ACTIVITY OF HUMAN NEUROTROPHIN-3 IN OLFACTORY ENSHEATHING CELLS MEDIATED BY RETROVIRAL VECTOR
YANG Hao JIN Wei Lin WANG Chun Ting YOU Si Wei JU Gong ( Institute of Neurosciences, the Fourth Military Medical Unrversity, Xi 'an 710032)
We successfully constructed a retrovirus expression vector pRev-TRE-NT3. The supernatant with highest retroviral liters was obtained by transfection of two retovirus vector, pRev-TRE-NT3 and pRev-Tet-On, into an ecotropic Ecopack-293 cells. Primarily cultured rat olfactory ensheathing cells (OECs) were co-injected 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 N'F-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. 78kb 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 cD-NA inserting pRev-TRE vector was integrated correctly; (2) NT-3-modified OEC culture supernatant contained 28kd N'F-3 which can bind NT-3 antibody in western-blot ting 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.
【Fund】： 国家自然科学基金(30070799);; 国家“973”课题项目“脑功能和脑重大疾病的基础研究子课题:脊髓损伤修复的基础研究”(G1999054009)
【CateGory Index】： Q42
【CateGory Index】： Q42