Expression of adenovirus-mediated glial cell line-derived neurotrophic factor gene in bone marrow stromal cells in vitro and its biological activity / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone marrow stromal cells(BMSCs) are the ideal gene target cells and will have a bright future in the gene therapy of spinal cord injury.OBJECTIVE:To detect the expression of glial cell line - derived neurotrophic factor(GDNF) gene after BMSCs were infected by adenovirus-medialed GDNF (Adv-GDNF) in vitro and to explore its biological activity.DESIGN: A randomized controlled trial study.SETTING: Laboratory of Orthopedic DepartmentMATERIALS The experiment was completed in the Laboratory of Orthopedic Department, Affiliated Tongji Hospital of Tong ji Meidcal College,Huazhong University of Science and Technology. Twenty-four SD rats of either gender, weighing (180 ± 20) g.INTERVENTIONS: BMSCs were infected by Adv-GDNF in vitro and then cocultured with spinal cord dorsal root ganglion. The three methods, immunofluorescent chemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay(ELISA) were used to evaluate GDNF expression in the BMSCs. The biological activity of GDNF was observed by a phase contrast microscope.MAIN OUTCOME MEASURES:Primary outcomes:①RT-PCR;②results of immunofluorescent chemical examination;③biological activity of GDNF in vitro. Secondary outcomes:①culturing and identification of BMSCs②time-effect relationship of GDNF expression revealed by ELISA.RESULTS: Immunofluorescence displayed expression of GDNF in BMSCs 48hours after Adv-GNDF infection. RT-PCR analysis demonstrated expression of GDNF mRNA 24 hours after Adv-GNDF infection. ELISA confirmed the presence of GDNF in the liquid supernatant of BMSCs 24 hours after Adv-GDNF infectionn and showed that GDNF was secreted. The supernatant can promote the neurite outgrowth in the rat dorsal root ganglion(DRG).CONCLUSION: It is demonstrated that BMSCs infected by Adv-GDNF can express GDNF steadily and the expressed GDNF has the activity of promoting neurite outgrowth, which lays a foundation of the GDNF gene therapy for spinal cord injury.