Effect of rat platelet-rich plasma gel on autologous adipose-derived mesenchymal stem cells overexpressing glia-derived neurotrophic factor
Objective To investigate the effect of rat platelet-rich plasma(PRP)gel on autologous adipose-derived mesenchymal stem cells(ADSCs)overexpressing glial-derived neurotrophic factor(GDNF).Methods This study was an experimental study.Five adult male Sprague-Dawley rats were used,and the primary ADSCs were obtained by collagenase digestion,and then the cells were identified successfully.The 3rd passage of ADSCs were obtained and divided into negative control group infected with unloaded adenovirus and overexpressing GDNF group infected with overexpressing GDNF adenovirus,according to random number table method(the grouping method was the same below).After 48 hours of culture,the infection of cells was observed.Five adult male Sprague-Dawley rats were used,and the PRP was obtained after collecting blood by differential centrifugation.PRP was prepared into a gel and its microstructure was observed by scanning electron microscope.The ADSCs of 3rd passage were added into the PRP solution mixture and cultured for 48 hours after gelation.The cell growth was observed by hematoxylin-eosin staining and calcein/propyl iodide staining.ADSCs infected with unloaded adenovirus and ADSCs infected with overexpressing GDNF adenovirus were routinely cultured in PRP gel.After 48 hours of culture,the cell growth was detected by calcein/propyl iodide staining.After culture for 24,48,72 hours and 1,2,3,4 weeks,the supernatant of cell culture medium was collected,the absorbance value was determined by microplate analyzer,and the GDNF content was calculated,with the sample number of 3.After 48 hours of culture,the expression of S100 protein(a specific marker of Schwann cells)was detected by immunofluorescence assay.Results After 48 hours of culture,the proportions of cells infected with adenovirus in negative control group and overexpressing GDNF group were close to 90％,and the cell growth was good.The cells in negative control group grew normally.The morphology of the cells in overexpressing GDNF group changed significantly with 80％-90％of the cells having two or more protrusions,and the protrusions were interwoven to form a network wherever the cells gathered.PRP gel formed a three-dimensional network structure with different pore sizes.After 48 hours of culture,ADSCs could be well attached to PRP gel,and 98％of the cells were alive.After 48 hours of culture,ADSCs infected with unloaded adenovirus grew well and showed typical ADSC-like spindle-shaped growth.ADSCs infected with overexpressing GDNF adenovirus grew well,and most of the cells had two or more protrusions,and the protrusions were interwoven into a network.After culture for 24,48,72 hours and 1,2,3,4 weeks,the content of GDNF in the supernatant of ADSCs infected with overexpressing GDNF adenovirus was(90±10),(133±15),(150±10),(137±15),(132±18),(120±10),and(127±16)μg/mL,which was significantly higher than(42±7),(44±7),(43±6),(47±6),(49±5),(49±6),and(51±4)μg/mL of ADSCs infected with unloaded adenovirus(with t values of 6.20,8.08,15.18,9.12,7.99,9.61,and 7.86,respectively,P＜0.05).After 48 hours of culture,the fluorescence intensity of S100 protein expression of ADSCs infected with overexpressing GDNF adenovirus was significantly stronger than that of ADSCs infected with unloaded adenovirus.Conclusions The prepared autologous three-dimensional PRP gel has good biocompatibility and can carry rat GDNF-overexpressing ADSCs and release GDNF slowly,inducing ADSCs to differentiate into Schwann cells that express high level of S100 protein.
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