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BACKGROUND:Mineralized nodules are the mature marker of osteoblast differentiation, and the observation methods mainly use alizarin red staining. OBJECTIVE:To compare the observation results of mineralized nodules by three methods, and to explore their characteristics and advantages, as wel as further application in the research of bone disease. METHODS:The rat osteoblast-like cellline UMR-106 were cultured in the fresh medium that was changed every day, for 14 days. Alizarin red staining-light microscope, tetracycline fluorescence labeling-laser confocal scanning microscopy, and scanning electron microscopy were used to observe mineralized nodules. The calcium content of mineralized nodules was quantified using scanning electron microscopy and energy dispersive spectroscopy. In addition, tumor necrosis factor alpha that could inhibit the proliferation and differentiation of osteoblasts was used as the control. RESULTS AND CONCLUSION:The three morphology methods could be used to observe the mineralized nodules of normal osteoblasts. As for tumor necrosis factor alpha, no mineralized nodules of osteoblasts were observed by alizarin red staining-light microscopy;smal mineralized nodules were observed by tetracycline staining-laser scanning confocal microscopy and scanning electron microscopy, suggesting tetracycline staining and scanning electron microscopy were more sensitive in the observation. Scanning electron microscopy could be used to observe the submicroscopic structures of mineralized nodules in the osteoblasts, and the formation of mineralized nodules, including the calcium secretion. Additional y, scanning electron microscopy combined with energy dispersive spectroscopy analysis can successful y quantify and position the mineralized nodules, indicating a potential application in the research of bone diseases.
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BACKGROUND:Tougu Xiaotong capsule is the clinical prescription for the treatment of osteoarthritis in Fujian University of Traditional Chinese Medicine, and the previous studies mainly focus on effect to cartilage. OBJECTIVE:To observe the effect of Tougu Xiaotong capsule on the proliferation and differentiation of osteoblasts as wel as the expressions of bone remodeling correlated factors. METHODS:Rat osteoblast-like cel line ROS17/2.8 cel s were incubated with Tougu Xiaotong capsule. The ROS17/2.8 cel s were divided into blank control group and Tougu Xiaotong capsule groups with different concentrations. The cel proliferation was determined by methylthiazolyldiphenyl-tetrazolium bromide assay. Osteoblast differentiation biomarkers alkaline phosphatase activity, osteocalcin and bone mineralized nodules were measured with colorimetry, enzyme-linked immunosorbent assay and alizarin red staining, respectively. The real-time PCR and enzyme-linked immunosorbent assay were used to detect the expressions of bone remodeling factors osteoprotegerin/receptor activator of nuclear factorκB ligand. RESULTS AND CONCLUSION:Compared with the control group, the Tougu Xiaotong capsule with the concentration of 0.25-2 g/L could significantly promote the ROS17/2.8 cel proliferation (Ppercentage of bone remodeling factors osteoprotegerin/receptor activator of nuclear factorκB ligand (Pproliferation and differentiation of osteoblasts and bone remodeling.
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Microscopic and histochemical methods were used to investigate flavonoids localization in the leaf and the stem of the Sarcandra glabra. The results indicated that flavonoids distributed mainly in epidermis, collenchyma, vascular bundles, secretory cells and palisade tissue of leaf. In the stem, they distributed mainly in epidermis, collenchyma, phloem and secretory cells. Histochemical localization of flavonoids using 5% solution of NaOH is convenient, rapid and reliable. The content of flavonoids in the leaf was higher those than in the stem. For sustainable utilization of the resources we suggested that only the leaves could be harvested.
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Flavonoids , Metabolism , Magnoliopsida , Metabolism , Microscopy , Plant Leaves , Metabolism , Plant Stems , MetabolismABSTRACT
Objective To study the effect of Jiedu-xiaozheng decoction on theUltrastructure changes and quantitative analysis of SGC-7901 in vitro and its mechanisms. Methods The compound prescription Jiedu-xiaozheng decoction was used to prepare serum containing drug. Serologic pharmacology methods was applied. The decoction was used to co-culture the gastric cells for24 h and 72 h, and the ultrastructure of tumor cells was observed by the transmission electron microscope. Then the parameters of the nucleus and mitochrondria were determined in SGC-7901 cells, including volume density(Vv) ,form factor PE( PE), average volume(V) ,length(L) average surrace area(S),contrasting to cytoplasma. The SIS powervision was applied to analyze their difference. Results Jiedu-xiaozheng decotion can result in reduced microvilli, deflated volume, decreased mitochondrion volume and reduced ridge. The volume density(Vv) ,form factor PE(PE) ,average volume(v) ,length (L) average surrace area(S) reduced in the 24 h group clearly in Jiedu-xiaozheng decoction group compared to control group. Conclusion Jiedu-xiaozheng decoction can significantly induce SGC-7901 cells apoptosis, exhibiting certain early ultrastructure changes such as marginated heterochromatin and mitochondrion pyknosis. The quantity analysis by electronic imaging system may objectively reveal the ultrastructural changes of cell nucleus and mitochondria.
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Objective To investigate the influence of pinealectomy(Px)and melatonin (MLT) supplementation on CD4~+CD25~+Treg cells development and Foxp3 expression in rat thymus. Methods One hundred and twenty clean-grade male SD rats were divided into five groups: normal control group, sham pinealectomized group, pinealectomized group, Px +7.5 mg/(kg·d) MLT group, Px +15 mg/(kg·d) MLT group, and the thymus were taken out at the 4th week and the 8th week;Flow cytometric analysis was used to analyse the number of double positive cells in the thymus and peripheral blood;Semiquantitative RT-PCR and immunohistochemistry were applied to analyse Foxp3 expression of rat thymus. Results There was no difference of the number of CD4~+CD25~+ Treg cells in rat thymus between Px and normal/sham group, and the number increased dependently on time and dose after MLT supplementation. In rat peripheral blood the double positive cells significantly increased in models of Px at the 4th week, and then backed to normal level after MLT supplementation. The results showed no significant changes in all groups at the 8th weeek;At the 4th weeks, Foxp3 expression increased in the thymus of Px rats compared to nomal/sham group, which returned to normal level after MLT supplementation. On the other hand, Foxp3 expression showed no significant difference in all groups at the 8th weeek.Conclusion Px made no difference between the development of CD4~+CD25~+Treg cells in rat thymus, but resulted in significant increase of thymic output and Foxp3 expression. All the effects disappeared at the 8th week. MLT supplementation could reverse the abnormity.
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Objective To study the effect of pinealctomy and melatonin on the cell cycle distribution during T cell development in thymus. Methods SD rats were divided into 5 groups:normal controls,sham-operated controls,pinealectomy,pinealectomy+melatonin injection 7.5 mg/kg; and pinealectomy+melatonin injection 15 mg/kg.The animals were sacrificed at 4 or 8 weeks after operation.cycilnA +and cyclinE +T cells in thymus were dyed with immunocytochemical ABC methods, measured by computer image analysis.Cell cycle analysis was performed by flow cytometry.The expression of cyclin-dependent kinase inhibitor 1? was tested by RT-PCR method. Results The effect of pinealectomy on the expression of cyclinE was stronger than that of cycilnA. Pinealectomy promotes the synthesis of cyclinE in thymus.The percentage of G-0/G-1 phase cells was decreased and G-2/M phase cells was increased.These would be convalescente by supplement with melatonin,but the reaction to melatonin was differencet in thymus cortex and in medulla.Melatonin enhanced the expression of cyclin-dependent kinase inhibitor 1? in thymus.Conclusion Pinealectomy influences notably the T cells development in thymus.Rejuvenation of degenerative thymus could be observed by melatonin administration.