The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×10 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1.
Polygonum chinense Linn. (Polygonum chinense L.) is one of the main raw materials of Chinese patent medicines such as Guangdong herbal tea. The increasing antibiotic resistance of S. aureus and the biofilm poses a serious health threat to humans, and there is an urgent need to provide new antimicrobial agents. As a traditional Chinese medicine, the antibacterial effect of Polygonum chinense L. has been reported, but the antibacterial mechanism of Polygonum chinense L.aqueous extract and its effect on biofilm have not been studied in great detail, which hinders its application as an effective antibacterial agent. In this study, the mechanism of action of Polygonum chinense L.aqueous extract on Staphylococcus aureus (S. aureus) and its biofilm was mainly evaluated by morphological observation, flow cytometry and laser confocal experiments. Our findings demonstrate that Polygonum chinense L.aqueous extract has a significant bacteriostatic effect on S. aureus. The result of growth curve exhibits that Polygonum chinense L.aqueous extract presents a significant inhibitory effect against S. aureus. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) reveals that Polygonum chinense L.aqueous extract exerts a potent destruction of the cell wall of S. aureus and a significant inhibitory effect on the formation of S. aureus biofilm. In addition, flow cytometry showed the ability of Polygonum chinense L.aqueous extract to promote apoptosis by disrupting cell membranes of S. aureus. Notably, confocal laser scanning microscopy (CLSM) images illustrated the ability of Polygonum chinense L.aqueous to inhibit the formation of S. aureus biofilms in a dose-dependent manner. These results suggested that Polygonum chinense L.aqueous is a promising alternative antibacterial and anti-biofilm agent for combating infections caused by planktonic and biofilm cells of S. aureus.
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