摘要
BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and myelin sheath repair. However, this effect remains very short-lived. OBJECTIVE： To transfect NT-3 into OECs and to observe the biological activity of OEC-expressing NT-3. DESIGN, TIME AND SETTING： This genetic engineering, in vitro experiment was performed in the Provincial Hospital Affiliated to Shandong University between January 2007 and October 2008. MATERIALS： Trizol Reagent kit was purchased from Gibco, USA; reverse transcription kit, NT-3 Emax ImmunoAssay System reagent was purchased from Promega, USA. METHODS： Neonatal Wistar rat OECs were established as primary cultures and were transfected with pN2A-NT-3 viral vector. The OECs with the highest virus titer and stable cellular growth served as the transfection group; OECs transfected with NT-3-free retrovirus carrier pN2A served as the empty vector group; un-transfected OECs served as the control group. After adherence, the logarithmically cultured PC12-TrkC cells were plated in OECs supernatant from the transfection and empty vector groups, as well as 20 μL PBS, and cultured for 4 days. MAIN OUTCOME MEASURES： NT-3 mRNA expression in OECs, fluorescence of NT-3-positive cells in the transfection group and control group; influence of OECs secreting NT-3 on the differentiation ratio of PC12-TrkC cells. RESULTS： NT-3 mRNA expression was observed 24 hours after transfection and lasted for 28 days which was greater than the control and empty vector groups （P 〈 0.01）. A large number of NT-3-positive cells were observed in the transfection group, and immunofluorescence was greater than the control and empty vector groups. PC12-TrkC cells co-cultured with OECs from the transfection group exhibited a thick and long cell process, increased cell density, and the differentiation ratio was increased （P 〈 0.01）. CONCLUSION： Recombinant double replica retrovirus NT-3 gene was stably and effectively expressed in OECs, and the expressed NT-3 possessed biological activity that promoted neuronal survival.
BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and myelin sheath repair. However, this effect remains very short-lived. OBJECTIVE： To transfect NT-3 into OECs and to observe the biological activity of OEC-expressing NT-3. DESIGN, TIME AND SETTING： This genetic engineering, in vitro experiment was performed in the Provincial Hospital Affiliated to Shandong University between January 2007 and October 2008. MATERIALS： Trizol Reagent kit was purchased from Gibco, USA; reverse transcription kit, NT-3 Emax ImmunoAssay System reagent was purchased from Promega, USA. METHODS： Neonatal Wistar rat OECs were established as primary cultures and were transfected with pN2A-NT-3 viral vector. The OECs with the highest virus titer and stable cellular growth served as the transfection group; OECs transfected with NT-3-free retrovirus carrier pN2A served as the empty vector group; un-transfected OECs served as the control group. After adherence, the logarithmically cultured PC12-TrkC cells were plated in OECs supernatant from the transfection and empty vector groups, as well as 20 μL PBS, and cultured for 4 days. MAIN OUTCOME MEASURES： NT-3 mRNA expression in OECs, fluorescence of NT-3-positive cells in the transfection group and control group; influence of OECs secreting NT-3 on the differentiation ratio of PC12-TrkC cells. RESULTS： NT-3 mRNA expression was observed 24 hours after transfection and lasted for 28 days which was greater than the control and empty vector groups （P 〈 0.01）. A large number of NT-3-positive cells were observed in the transfection group, and immunofluorescence was greater than the control and empty vector groups. PC12-TrkC cells co-cultured with OECs from the transfection group exhibited a thick and long cell process, increased cell density, and the differentiation ratio was increased （P 〈 0.01）. CONCLUSION： Recombinant double replica retrovirus NT-3 gene was stably and effectively expressed in OECs, and the expressed NT-3 possessed biological activity that promoted neuronal survival.
基金
the National Natural Science Foundation of China,No.30770751
the Foundation for Youth of Shandong Bureau of Public Health,No.2007QZ002
the Doctoral Foundation of Shandong Scientific and Technological Bureau,No. 2008BS03004
