ABSTRACT
Objective: To investigate the effects of polyactic-co-glycolic (PLGA) electrospinning membrane carrying paclitaxel liposomes with different concentrations on the proliferation and differentiation of the neural stem cells (NSCs) â™ and to evaluate the feasibility of PLGA electrospinning membrane carrying paclitaxel liposomes in the tissue engineering repair of spinal cord injury. Methods: The paclitaxel liposomes and PLGA were mixed in different proportions and the electrospinning technique was used to construct 1, 5 and 10 ng • L electrospinning membrane carrying paclitaxel liposomes. The NSCs were isolated and purified from the fetal rat brain tissue. The diameters of PLGA electrospinning membrane carrying paclitaxel-liposomes in various groups were detected by scanning electron microscope (SEM) â™ and the isolated and purified NSCs were identified by immunofluorescence staining. The NSCs were cultured in PLGA electrospinning membrane carrying paclitaxel-liposomes with different concentrations (0, 1, 5 and 10 p. g • L ) as PLGA electrospinning membrane group and PLGA electrospinning membrane carrying 1, 5 and 10 n g • L paclitaxel-liposomes groups. The proliferation levels of the NSCs in various groups was detected by MTT method, and the expression levels of Tuj-1 and GFAP mRNA in the NSCs in various groups were detected by RT-PCR method. Results: The PIjGA electrospinning membrane carrying paclitaxel-liposomes were white thin films. The SEM results showed that there was no statistical differences in the fiber diameters of PLGA electrospinning membrane carrying paclitaxel-liposomes between various groups ( P > 0.05). The immunofluorescence staining results showed that the cells isolated from fetal rat brain tissue were spherical and positive expression of Nestin protein. The MTT assay results showed that the proliferation level of NSCs in PLGA electrospinning membrane carrying 5 fxg ' L paclitaxel liposomes group was higher than those in the other three groups (P<"0.05). The RT-PCR results showed that compared with PLGA electrospinning membrane group, the expression level of Tuj-1 mRNA in the NSCs in PLGA electrospinning membrane carrying 5 pg ∗ L paclitaxel-liposomes group was increased ( P< 0. 05) and the expression level of GFAP mRNA in the NSCs in PLGA electrospinning membrane carrying 5/ig ∗ L paclitaxel-liposomes group was decreased ( P < 0.05 ). Conclusion: Medium concentration of paclitaxel can promote the proliferation of NSCs and induce the differentiation of NSCs into the neurons. PIjGA electrospinning membrane carrying medium concentration of paclitaxel-liposomes has certain application value in the repair of spinal cord injury.
ABSTRACT
Objective To investigate the effect of polylactic-co-glycolic acid (PLGA)/graphene oxide (GO) nanofibers combined with brain derived neurotrophic factor (BDNF) on neural stem cells (NSCs) proliferation and differentiation as well as on the spinal cord injury repair.Methods PLGA/GO nanofibers were manufactured and absorbed with BDNF,and the microstructure of PLGA/GO nanofibers was observed by scanning electron microscope.The loading efficiency and release curve of BDNF on PLGA/GO nanofibers were measured by ELISA.NSCs were implanted on the surface of PLGA/GO and PLGA/GO/BDNF nanofibers.The absorbance values of each group were measured by MTT method,and the expression of Tuj-1 was observed by immunofluorescence and PCR.A total of 30 female SD rats were divided into control group (n =10),PLGA/GO group (n =10) and PLGA/GO/BDNF group (n =10) according to random number table.T9 spinal cord tissue was cut by Venus scissors to establish spinal cord hemisection injury model of rats.PLGA/GO and PLGA/GO/BDNF nanofibers were implanted onto the surface of injury site.BBB score was used to assess the motion functional recovery of the rats at 1,7,14 and 28 days after operation.Immunofluorescence staining of neuron specific nucleoprotein (NeuN)and glial fibrillary acidic protein (GFAP) were performed to observe the expressions of neurons and astrocytes at the injured site respectively one month after injury.Results The PLGA/GO nanofibers showed an irregular smooth fiber-like structure,and the average fiber diameter was (987.5 ± 176.3)nm.NSCs could differentiate into neurons on the nanofibers.The result of ELISA showed loading rate of BDNF on PLGA/GO nanofibers was about 47.5％.The release curve showed that BDNF was first released about 30％ on the first day and then about 60％ on the 21st day.The results of MTT and PCR showed that optical density value and Tuj-1 gene expression in the PLGA/GO/BDNF group were significantly higher than those in the PLGA/GO group (P ＜ 0.05).The animal experiment results showed that the BBB score of PLGA/GO/BDNF group was (15.3 ±0.7) points at 28 days after injury,which was significantly higher than that of the injury control group [(11.8 ± 0.8) points] and that of PLGA/GO group [(12.7 ±0.8) points] (P ＜ 0.05).Immunofluorescence results showed that the expression of NeuN in PLGA/GO/BDNF group was 13.7 ± 2.2,significantly higher than that in injury control group (4.3 ± 2.9) (P ＜0.05),and the expression of GFAP in PLGA/GO group was (25.6 ± 4.3) ％ significantly lower than that in injury control group [(38.5 ± 6.2) ％] and PLGA/GO group [(36.7 ± 7.3) ％] (P ＜ 0.05).Conclusion PLGMGO nanofibers combined with BDNF can effectively promote the proliferation and neuron differentiation of NSCs in vitro and repair spinal cord injury in vivo through orthotopic transplantation at the injury site.