ABSTRACT
<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.</p>
Subject(s)
Animals , Rats , Acetylcysteine , Pharmacology , Bone Marrow Cells , Cell Biology , Metabolism , Cells, Cultured , Culture Media, Conditioned , Metabolism , DNA, Recombinant , Genetic Therapy , Methods , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor , Genetics , Metabolism , Lentivirus , Genetics , Neurons , Neuroprotective Agents , Metabolism , PC12 Cells , Plasmids , Genetics , Protease Inhibitors , Pharmacology , RNA, Messenger , Rats, Sprague-Dawley , Stromal Cells , Transduction, Genetic , MethodsABSTRACT
<p><b>OBJECT</b>To investigate the therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells (RPE-M) transplantation on rat model of Parkinson's disease (PD).</p><p><b>METHODS</b>Primary porcine RPE cells were harvested by enzyme digestion and expanded in culture medium. Determine the levels of dopamine (DA) and homovanillic acid (HVA) by high performance liquid chromatography electrochemical (HPLC) assay, and the levels of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were detected by ELISA. Alginate-polylysine-alginate (APA) microencapsulated cells were produced by using a high voltage electrostatic system. PD rat model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB). After that, the RPE-M was transplanted into the corpus striatum of PD rat, and then the rotation test scores were recorded and biochemical changes of the corpus striatum were tested.</p><p><b>RESULTS</b>The levels of DA, HVA, BDNF and GDNF secreted by RPE were stable in the RPE culture supernatant and were not changed by the microencapsulation. Eighty-three percent rats developed PD by unilateral lesion of 6-OHDA in the MFB. The RPE-M transplantation had therapeutic effect on 33% PD rats.</p><p><b>CONCLUSION</b>Porcine RPE cells grow actively in vitro and could secrete DA, HVA, BDNF, and GDNF constantly, which does not be affected by the passage culture and the APA miroencapsulation. RPE-M transplantation of may be a curative therapy for PD.</p>