Effect of glial cell-derived neurotrophic factor-modified adipose-derived stem cells on survival of co-cultured dopaminergic neurons / 中国组织工程研究

ABSTRACT

BACKGROUND: Reports regarding adipose-derived stern cells (ADSCs) differentiation into dopaminergic (DN) neurons are few in addition, there is not experimental evidence of the effect of ADSCs on maintaining the survival of DN neurons.OBJECTIVE: To investigate the effect of glial cell-derived neurokophic factor (GDNF)-modified adipose-derived stem cells on survival of DN neurons under co-cultured condition.DESIGN, TIME AND SETrlNG The in vitro cytology experiment was conducted at the Institute of Otolaryngology-Head and Neck Surgery and Key Laboratory of Zoonoses of Ministry of Education between March and December 2007.MATERIALS Wistar rats with 3-weeks-old, or 14 days of pregnancy were provided by Norman Bethune College of Medicine, Jilin University.METHODS: The GDNF recombinant adenovirus was constructed by using pAdTrackCMV and pAdEasy-1 system. DN neurons were obtained from the rostral mesencaphalic tegmentum of Wistar rat embryos by using trypsin and collagenase method. ADSCs isolated from rat inguinal fat pads were digested with collagenase Ⅱ, cultured and passaged in vitro. When the cells reached 60% cenfluency at the 3rd passage, cells were transfectad with 1×109vp/mL of Ad-GDNF for 1 hour and then transferred into growth medium for another 24 hours, and GDNF level in cell supematant was detected by ELISA assay. Meanwhile, the co-cultured of ADSCs and DN neurons were carried out for following 7 days. With GFP-modified ADSCs was served as a control group.MAIN OUTCOME MEASURES: The effect of co-cultured condition on the survival of DN neurons, as well as the differentiation of GDNF-modified ADSCs was detected by immunofluorescence staining.RESULTS: GDNF appeared in ADSCs supematant at 24 hours after Ad-GDNF transfection and reached a peak at 72 hours.There was approximately 80% GFP-positive labeled in ADSCs. The tyrosinase hydroxylase staining results demonstrated that the rate of survival DN neurons were significantly increased than in DA neurons cultured alone, co-cultured group of GFP-modified ADSCs and GDNF-modified ADSCs groups (55%, 15%, 25%, P < 0.01). However, there were no co-expressing TH and GFP positive cells appeared at 7 days of co-culture, which indicated that the co-cultured condition was not available to ADSCs differentiation.CONCLUSION: The co-cultured of GDNF modified ADSCs and DN neurons can promote the survival and growth of cultured DN neurons, however, it can not induce ADSCs differentiate into DN neurons.