RESUMO
Objective: To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro. Methods: NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% ( W/ V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D. Results: The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group ( P0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B ( P0.05). Conclusion: NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.
RESUMO
Objective To determine whether macrophages can behave as antigen presenting cells participating the formation of immunological synapse in rheumatoid arthritis (RA) and whether this process can affect the apoptosis. Moreover, this study was aimed to observe the function of cyclophilin A (CypA) in immunological synapse formation and its role in regulating the apoptosis of macrophages. Methods human acute monocytic leukemia cell line (THP-1) induced macrophages were coated with staphylococcal enterotoxin B(SEB) (100 ng/ml) and co-cultured with activated Jurkat T cells (human acute T-cell leukemia cell line), then incubated in the RPMI-1640 for 16 hours to induce apoptosis. The apoptosis of the macrophages were analyzed by flow cytometry by Annexin V-PI staining. The macrophages cultured in the RPMI-1640 alone were used as control. Meanwhile, CypA (200 ng/ml) were added to or not added in order to observe the apoptosis of macrophages. The function of CypA and the apoptosis of macrophages isolated from RA peripheral blood were also investigated through co-culture with CD4+T cells isolated by immunomagnetic beads. Comparisons between groups were performed by two-sample t-tests. Results In the peripheral blood of healthy people and RA patients, the apoptosis of macrophages which participated immunological synapse was (32.9±2.8)%, (24.7±1.6)%, (14.5±1.2)% respectively, which was significantly lower than the apoptosis of macrophages cultured alone [ respectively for (61.4±2.4)%, (45.5±2.6)%, (22.9±1.5)%, (P<0.05) ]. After CypA was added, the apoptosis of macrophages in cell lines, healthy people and RA patients decreased to (27.2±2.1)%, (20.1±1.1)%, (12.9±1.0)%, lower than the apoptosis of macrophages which participated immunological synapse formation (P<0.05). Conclusion In RA, the macrophages participate in the formation of immunological synapse by interacting with CD4+ T cells. They can significantly reduce the apoptosis on themselves. CypA can enhance this effect. These results provide a new theoretical foundation for prolonged survival of macrophages in RA, which can secrete a variety of cytokines to enhance inflammation and joint destruction.
