羟基脲对大鼠Sertoli-germ 细胞共培养的影响

目的:观察羟基脲(Hydroxyurea, HU) 对雄性大鼠Sertoli-germ 细胞共培养的细胞毒性作用。方法:在Sertoli-germ 细胞共培养模型中分别加入不同浓度HU(0、 10^-3、 10^-2、 10^-1 mol/L) 后, 孵育96 h; 以及加入10^-2 mol/L HU 后分别孵育24、 48、 72、 96 h 。结果:在各给药组, 96 h 时HU 导致germ细胞从Sertoli 细胞层的脱落增加, 呈现量效关系。在10^-3 mol/L 以上浓度组, 细胞活性逐渐下降, 细胞脱落增加。同样, 随着给药时间的增加, germ细胞脱落率和乳酸脱氢酶(LDH) 的漏出逐渐增加, 细胞活性逐渐下降。结论:HU 对雄性大鼠睾丸Sertoli-germ 细胞共培养模型的germ 细胞具有明显毒性作用。     

Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells

Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the Ussue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding （i） critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and （ii） the usage of monocultures in clinical application in BTE.     

Metabolomic profile of milk fermented with Streptococcus thermophilus cocultured with Bifidobacterium animalis ssp. lactis,Lactiplantibacillus plantarum,or both during storage

In this study, the microbial interactions among cocultures of Streptococcus thermophilus (St) with potential probiotics of Bifidobacterium animalis ssp. lactis (Ba) and Lactiplantibacillus plantarum (Lp) in fermented milk were investigated during a storage period of 21 d at 4°C, in terms of acidifying activity (pH and titratable acidity), viable counts, and metabolites. A nontargeted metabolomics approach based on ultra-high-performance liquid chromatography coupled with mass spectrometry was employed for mapping the global metabolite profiles of fermented milk. Probiotic strains cocultured with St accelerated milk acidification, and improved the microbial viability compared with the single culture of St. The St–Ba/Lp treatment manifested a higher bacteria viability and acidification ability in comparison with the St–Ba or the St–Lp treatment. Relative quantitation of 179 significant metabolites was identified, including nucleosides, AA, short peptides, organic acids, lipid derivatives, carbohydrates, carbonyl compounds, and compounds related to energy metabolism. The principal component analysis indicated that St treatment and coculture treatments displayed a complete distinction in metabolite profiles, and Lp had a larger effect than Ba on metabolic profiles of fermented milk produced by cofermentation with St during storage. The heat map in combination with hierarchical cluster analysis showed that the abundance of metabolites significantly varied with the starter cultures over the storage, and high abundance of metabolites was observed in either St or coculture samples. The St-Ba/Lp treatment showed relatively high abundance for the vast majority of metabolites. These findings suggest that the profile of the metabolites characterizing fermented milk samples may depend on the starter cultures, and incorporation of probiotics may considerably influence the metabolomic activities of fermented milks.     

Ethanologens vs. acetogens: Environmental impacts of two ethanol fermentation pathways

Bioconversion production of ethanol from cellulosic feedstock is generally proposed to use direct fermentation of sugars to ethanol. Another potential route for ethanol production is fermentation of sugars to acetic acid followed by hydrogenation to convert the acetic acid into ethanol. The advantage of the acetogen pathway is an increased ethanol yield; however, using an acetogen requires the additional hydrogenation, which could substantially affect the life cycle global warming potential of the process. Assuming a poplar feedstock, a cradle to grave Life cycle assessment (LCA) is used to evaluate the environmental impacts of an acetogen based fermentation pathway. An LCA of a fermentation pathway that uses ethanologen fermentation is developed for comparison. It is found that the ethanologen and acetogen pathways have Global Warming Potentials (GWP) that are 92% and 46% lower than the GWP of gasoline, respectively. When the absolute GWP reduction compared to gasoline is calculated using a unit of land basis, the benefit of the higher ethanol yield using the acetogen is observed as the two pathways achieve similar GWP savings. The higher ethanol yield in the acetogen process plays a crucial role in choosing a lignocellulosic ethanol production method if land is a limited resource.     

A keratinocytes-melanocytes coculture system for the evaluation of active ingredients' effects on UV-induced melanogenesis

A new experimental design, more reliable for in vitro testing of active ingredients' effect on ultraviolet (UV)-induced melanogenesis has been carried out. It uses a bicompartmental coculture system where cell communication between keratinocytes and melanocytes can take place. Thus, this experimental situation enables to monitor the effect of biological agents released by both cell types on melanogenesis and the interference of tested compounds with this 'paracrine linkage'. Experiments with UVB-irradiated cocultures show the importance of cell communication in the melanogenic response. In this model, the endogenous mediator, nitric oxide (NO), increased melanin production. Different compounds were tested in the coculture system, and comparison with data obtained from irradiated monocultures of melanocytes enables to distinguish a specific effect on cell communication. In addition, this more close-to-reality experimental model proved to provide a valuable first approach for the assessment of the 'bioavailability' of the tested substances. Finally, the effect of an innovative photoprotective agent capable of 'boosting' UV-induced melanogenic cell communication is presented.     

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.