ABSTRACT
OBJECTIVE@#To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs.@*METHODS@#MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated.@*RESULTS@#ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs.@*CONCLUSION@#In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.
Subject(s)
Humans , Autophagy , Mesenchymal Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Spondylitis, Ankylosing , TOR Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of a microencapsule scaffold capable of sustained release of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) in promoting the osteogenic differentiation of rat periosteum-derived stem cells (PDSCs) in vitro.</p><p><b>METHODS</b>PDSCs from 4-week-old SD rats, after identification of the surface markers using flow cytometry, were induced to differentiate into osteoblast, chondroblast, and adipocyte lineages. The differentiated cells were verified by staining with Alizarin red, toluidine blue, alcian blue, oil red O and by immunofluorescence assay. FGF-2/PELA/BMP-2, FGF-2/PELA, PELA/BMP-2 and PELA microcapsules were prepared, examined for surface morphologies using scanning electron microscopy (SEM), and tested for controlled release of FGF-2 and BMP-2 using ELISA. The third passage of PDSCs were cultured in the presence of the aqueous extracts of one of the 4 materials, and alkaline phosphatase (AKP) activity in the culture media was detected at 7 and 14 days of culture; the expression levels of osteogenesis-related genes were quantified with quantitative real-time PCR (qRT-PCR). The osteogenic differentiation ability of the PDSCs cultured with the extracts was compared.</p><p><b>RESULTS</b>The PDSCs, which expressed mesenchymal stem cell surface markers, were shown to have osteogenic, chondrogenic and adipogenic differentiation potentials. The cells cultured with the extract of FGF-2/PELA/BMP-2 microcapsules showed the highest AKP activity at 7 and 14 days of culture, and their expression levels of OCN and RunX-2 mRNA were the highest among the 4 groups; RunX-2 expression reached its peak level on day 14, and OCN mRNA expression level increased progressively as the culture time extended.</p><p><b>CONCLUSION</b>FGF-2/PELA/BMP-2 biomimetic controlled release microcapsules preserve the cytokine activities and are capable of promoting the osteogenic differentiation of rat PDSCs.</p>
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OBJECTIVE: To establish a method for predicting tablet hardness by near infrared diffuse reflection spectroscopy. METHODS: Tablet hardness value was obtained by hardness meter. Calibration model between NIR spectra and the hardness was establish by partial least squares regression (PLSR) method and BP-ANN method. RESULTS: Correlation coefficients (r), root mean squares error of cross-validation (RMSECV), and root mean square error of prediction (RMSEP) obtained by PLSR model were 0.977 8, 0.742 and 0.815 kg respectively. And the correlation coefficients of training set, monitor set and testing set by BP-ANN were 0.987 3, 0.985 6, and 0.986 8, with RSE% of 6.83%, 8.77%, and 6.69%, respectively. CONCLUSION: The prediction accuracy of BP-ANN nonlinear model is superior to the PLSR model.
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OBJECTIVE: To establish a rapid and nondestructive method to determine coating thickness of the total saponin of Radix Bupleuri enteric coated tablets by near infrared spectroscopy (NIRS) analytical technique. METHODS: Spectral pretreating methods, wavenumber selection and factors of NIRS were discussed in detail. Different modeling methods were compared and the methodology of model was investigated. RESULTS: Partial least squares regression (PLSR) was used for building the quantitative calibration model. Correlation coefficients(r), root-mean-squares error of cross-validation (RMSECV), and root mean square error of prediction (RMSEP) obtained by PLSR model were 0.9915, 3.6 and 3.24 μm respectively. CONCLUSION: Predicted results show that the established method is rapid, nondestructive, and reliable, which can be applied to the online monitor of Chinese medicine tablets coating process.