ABSTRACT
BACKGROUND:The intracel ular accumulation of reactive oxygen species leads to oxidative stress. Hypoxia is widespread in physiological and pathological condition. Variation of bone proliferation and differentiation when bone tissues cultured or bone cel s induced toxicity by reactive oxygen species under hypoxia have not yet been reported. OBJECTIVE:To observe the biological characteristics of MC3T3-E1 pretreated with different concentrations of hydrogen peroxide (H2O2) in hypoxia, thus understanding the cel mechanism underlying prolonged bone healing in the elderly with osteoporosis and diabetes. METHODS:The MC3T3-E1 cel s pretreated with different concentrations of H2O2 were cultured in different oxygen concentrations. The proliferation of MC3T3-E1 was detected by cel counting kit-8. The cel differentiation was detected through alkaline phosphatase staining and alizarin red staining. Total RNAs were extracted and used for analyzing the mRNA levels of col age type 1, alkaline phosphatase and Cbfa1. RESULTS AND CONCLUSION:When MC3T3-E1 pretreated with 200μmol/L H2O2 for 6 hours, the cel proliferation was increased with time, but lower than that in the control group. The alkaline phosphatase activity was weakened, and the number of mineralized nodes was decreased at the early stage of differentiation. When MC3T3-E1 pretreated with 400μmol/L H2O2 for 6 hours, the cel proliferation was decreased obviously. The alkaline phosphatase activity was stil weakened, and the number of mineralized nodes was decreased further, but not affected by hypoxia. When MC3T3-E1 pretreated with 400μmol/L H2O2 for 6 hours and then cultured in hypoxia, the mRNA expression of Cbfa1 was decreased, but the mRNA expressions of col age type 1 and alkaline phosphatase were significantly increased. These results suggest that MC3T3-E1 pretreated with low concentration of H2O2 show a significant decrease in proliferation, while MC3T3-E1 pretreated with a high concentration of H2O2 and cultured in hypoxia show a decrease in osteogenic differentiation, especial y at the early stage of alkaline phosphatase formation.
ABSTRACT
Objective To identify the mutations of DNA gyrase gyrA and Topoisomerase Ⅳ parC genes in quinolone-resistant Shigella flexneri clinical isolates and evaluate the relevance of amino acid changes in GyrA and ParC to quinolone resistance. Methods Based on the antimicrobial susceptibility testing, 47 S.flexneri clinical isolates with different quinolone susceptibility were selected and the fragments including the quinolone resistance-determining region (QRDR) in gyrA and parC were PCR amplified and sequenced. SAS (V 8.2) software was used to analyze the correlation between quinolone resistance and changes in GyrA and ParC. Results Sense mutation(s) in gyrA and parC were commonly observed in all of 44 quinolone-resistant isolates, whereas no sense mutation was found in the 3 quinolone-susceptible ones. The most frequent mutation is at codon 83(TCG→TTG) of gyrA, which was observed in 43 quinolone-resistant isolates. The mixed model analysis indicated that the alterations in amino acid 83 of GyrA and amino acid 80 of ParC are close related to nalidixic acid resistance (P