EXPRESSION OF HUMAN CNTF MEDIATED BY RETROVIRAL VECTOR IN OLFACTORY ENSHEATHING CELLS in vitro AND EFFECTS ON NEURONAL SURVIVAL AND NEURITE OUTGROWTH / 解剖学报

ABSTRACT

Objective To explore the effects of expressing human ciliary neurotrophic factor (hCNTF) mediated by retroviral vector in olfactory ensheathing cells(OECs) on the survival and neurite outgrowth of cultured neurons. Methods S\|hCNTF fragment was digested with endonucleases(Kpn I and Xba I) from pcDNA\-3\|S\|hCNTF plasmid and cloned into pRev\|TRE vector.The harvested pRev\|TRE\|hCNTF was identified and transfected with pRev\|Tet\|On into ecotropic Ecopack\|293 cells,resulting in 2 retroviral supernatants(pRev\|TRE\|hCNTF and pRev\|Tet\|On).Primarily cultured rat olfactory ensheathing cells(OECs) were co\|infected with the 2 retroviruses,and induced to secrete hCNTF with different concentrations of doxycline.The secreted hCNTF in OEC culture supernatant was detected with Western\|blot.Dorsal root ganglion (DRG) from a postnatal rat of 2 days was co\|cultured with CNTF\|modified OECs,and the supernatant was used to culture retinal ganglion cells(RGCs).Following ?\|tubulin immunocytochemical staining,the length of DRG neurites were measured,while the numbers of surviving RGCs were counted. Results 1.Individual 630bp and 400bp fragments were digested from pRev\|TRE\|S\|hCNTF expression vector with endonucleases(Hind Ⅲ and BamH Ⅰ),and respected direction and integration of hCNTF cDNA which inserted pRev\|TRE vector were identified; 2.The expression of 24kD CNTF proteins in CNTF\|modified OEC culture supernatant was positively\|correlated with the concentration of doxycline,while no such protein expression was detected in the control groups; 3.The number of surviving RGCs in CNTF\|modified OECs group(41^34?5^4) was significantly higher than those in unmodified OEC(23^15?4^7),OECs(24^55?5^8) and blank(16^8?6^5) groups;and 4^The neurites of DRG were longer (660?67?m) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418?45?m),Mock+OECs(400?65?m) and blank (0?m) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be expressed in OECs with a doxycline concentration\|dependent manner after transfected via pRev\|TRE\|S\|hCNTF vector,and possesses a marked enhancing effect on the survival and neurite outgrowth of cultured neurons.[