OBJECTIVE: To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotrophic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells (BMSCs). METHODS: pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. RESULTS: Virus stock of GDNF was harvested with the titer of 5.6 x 100,000 TU/mL. After transduction, GDNF-BMSCs successfully secreted GDNF to supernatant with higher concentration (800 pg/mL) than BMSCs did (less than 100 pg/mL). The supernatant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin (10 micromol/L). CONCLUSION: Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
OBJECTIVE: To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotrophic factor (GDNF), and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells (BMSCs). METHODS: pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. RESULTS: Virus stock of GDNF was harvested with the titer of 5.6 x 100,000 TU/mL. After transduction, GDNF-BMSCs successfully secreted GDNF to supernatant with higher concentration (800 pg/mL) than BMSCs did (less than 100 pg/mL). The supernatant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin (10 micromol/L). CONCLUSION: Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
Authors: T Kitada; S Asakawa; N Hattori; H Matsumine; Y Yamamura; S Minoshima; M Yokochi; Y Mizuno; N Shimizu Journal: Nature Date: 1998-04-09 Impact factor: 49.962