Proteomic analysis of chondromodulin-I-induced differentiation of mesenchymal stem cells into chondrocytes.

To identify novel proteins that might help clarify the molecular mechanisms underlying chondromodulin-I (ChM-I) induction of mesenchymal stem cells (MSCs) differentiate into chondrocytes. MSCs are triggered to differentiate into chondrocytes, which are recognized as important factors in cartilage tissue engineering. ChM-I is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. In this study, the proteomic approach was used to evaluate protein changes between undifferentiated MSCs and ChM-I-transfected MSCs. The expression of the protein spots was analyzed using two-dimensional gel electrophoresis. Then, 14 protein spots were identified between MSCs and ChM-I-transfected MSCs. 309 proteins were identified using mass spectrometry (MS). The differentially regulated proteins were categorized and annotated using Protein Analysis Through Evolutionary Relationships (PANTHER) analysis with the aid of the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. These proteins are included in a variety of metabolic pathways and signal transduction pathways, such as focal adhesion, glycolysis, actin cytoskeleton regulation, and ribosome. These results demonstrate novel information about the molecular mechanism by which ChM-I induce MSCs to differentiate into chondrocytes. These results also provide a solid foundation for the development of tissue-engineered cartilage.

Using bovine pituitary extract to increase proliferation of keratocytes and maintain their phenotype in vitro.

AIM: To investigate the effects of bovine pituitary extract on the proliferation of keratocytes and maintaining the keratocyte phenotype in vitro. METHODS: Single keratocytes were isolated by enzyme digestion for in vitro culture. Three groups were designed according to the different culture media: a bovine pituitary extract (BPE) group, a fetal bovine serum (FBS) group and the control group. The phenotypes and proliferation of cultured cells were evaluated by morphology, immunofluorescent staining and mRNA expression of CD34, Lumican, VSX1, α-SMA and proliferating cell nuclear antigen (PCNA). In the BPE group, cells underwent serial subcultivation, and their phenotypes were identified by immunofluorescent staining. To analyze the proliferation of keratocytes in different concentrations of BPE, six different concentrations were designed to ascertain the most appropriate amount. RESULTS: In the BPE group, the cells spread out and presented dendritic morphology, and their dendrites connected to one another to form networks. On the third passage, most cells maintained their phenotype. In the FBS group, the cells exhibited a dendritic appearance in early cultured stages, but their morphology subsequently changed into a fibroblast-like shape. The number of dendritic cells in BPE group was more than FBS and control groups. Immunofluorescent staining and real-time polymerase chain reaction (PCR) confirmed that few keratocytes underwent fibroblastic transformation in the BPE and control groups, and that proliferation was higher in the BPE group than in the control group. Although the proliferation was higher in the FBS group, many keratocytes underwent fibroblastic transformation. The analysis of cell morphology and mRNA expressions of CD34, PCNA and VSX1 in six group showed that different concentrations of BPE affected the proliferation obviously but didn't affect the keratocyte phenotype, and the concentration of 40µg/mL was the most appropriate one. CONCLUSION: BPE can improve the proliferation of keratocytes and maintain their phenotype in vitro. Many keratocytes can be harvested rapidly and provide seeds for the construction of corneal stroma.

[Comparison of the activities of silver nitrate with those of three antifungal agents against ocular pathogenic fungi in vitro].

OBJECTIVE: To investigate antifungal activity of silver nitrate compared with fluconazole, ketoconazole and amphotericin B against ocular pathogenic fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates (15 genera and 29 species) of ocular pathogenic fungi by broth dilution antifungal susceptibility testing of filamentous fungi (M38-A) approved by National Committee for Clinical Laboratory Standards (NCCLS). Final concentrations ranged from 0.031 to 16.000 mg/L for silver nitrate, ketoconazole and amphotericin B, from 0.5 - 256.0 mg/L for fluconazole. Minimum inhibitory concentration (MIC) was defined as the lowest drug concentration that showed absence of growth or complete growth inhibition (100%). The end points were determined as 100% growth inhibition for silver nitrate and amphotericin B, and > or = 75% growth inhibition for ketoconazole and fluconazole. RESULTS: The MICs at which 90% of isolates were inhibited (MIC(90)) of silver nitrate, ketoconazole, amphotericin B and fluconazole were 2.000, 512.000, 32.000 and 2.000 mg/L for Fusarium species, respectively; 1.000, 256.000, 2.000 and 2.000 mg/L for Aspergillus species, respectively; 2.000, 128.000, 4.000 and 2.000 mg/L for Alternaria alternate, respectively; 2.000, 4.000, 0.125 and 0.500 mg/L for Curvularia lunata, respectively; and 1.000, 256.000, 1.000 and 1.000 mg/L for unusual ocular pathogens, respectively. Silver nitrate was highly active against Aspergillus species (92.9% susceptible at a MIC of < or = 1.0 mg/L) and Fusarium species (96.3% susceptible at a MIC of < or = 2.0 mg/L). 95.6% of Fusarium species and 90.8% of Aspergillus species exhibited resistance to fluconazole, 44.1% of Fusarium species and 42.9% of Aspergillus species exhibited resistance to amphotericin B, 66.2% of Fusarium species exhibited resistance to ketoconazole. The activity of silver nitrate against the fluconazole-resistant, ketoconazole-resistant and amphotericin B-resistant strains was high. CONCLUSION: Silver nitrate has promising activity against a wide variety of ocular pathogenic fungi in vitro, and may have a role in future studies of antifungal eye drops and treating fungal keratitis.