Studies on development of serum-free conditioned media using Vero cells and DMEM with controlled concentration of glucose and pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine in vitro development of early preimplantation mouse embryos in various kind of serum-free conditioned media (SF-VCM) manufactured from DMEM cultured with Vero Cells. METHODS: A total of 846 two-cell mouse embryos were cultured in different kind of SF-VCM. SF-VCMs were divided into SF-VCM-10, -30 and -50 by media volume using DMEM #1 media, and divided into SF-VCM #1, #2 and #3 by controlled concentration of glucose and pyruvate (manufactured by DMEM #1: mixed three volume of DMEM-G (DMEM with glutamine without glucose and pyruvate) and one volume of DMEM-GGP (DMEM with glutamine, glucose, pyruvate), #2: mixed same volume of DMEM-G and DMEM-GGP and #3: mixed one volume of DMEM-G and three volume of DMEM-GGP, respectively). Experimental groups were mainly added 10% SSS, and 20% hFF was added to only Control group co-cultured with Vero cells. Development of embryos was observed every 24 hours. Results between different groups were analyzed using Chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: In vitro developmental rate by each cleavage stages of mouse embryos cultured in SF-VCMs with a various volumes were significantly (P<0.05) higher in SF-VCM-30 (morula< or =: 97.2%, Blastocyst (BL)< or =: 97.2%, Hatching BL< or =: 82.2%) than other groups. In the rate of development on in vitro co-culture vs. a various SF-VCMs manufactured by DMEM controlled concentration of glucose and pyruvate, Group I (SF-VCM #1) was higher than other groups in each cleavage stages (morula< or =: 98.1%, Blastocyst (BL)< or =: 97.1%, hatching BL< or =: 81.7%, respectively). Moreover, specially, in the developmental rate into the hatching blastocyst < or = after 96 hours in vitro culture, Group I (81.7%) was significantly higher than control group (67.6%, P<0.05). CONCLUSION: SF-VCM #1 manufactured by volume of 30 mL DMEM #1 media cultured in vitro for 48 hours in 250 mL flask was the most effective on in vitro developmental rate of mouse preimplantation embryos. Therefore, it is expected that SF-VCM #1 has application to human IVF-ET.

The binding affinity of glucocorticoid receptor in the peripheral blood mononuclear cells from patients with steroid-resistant and steroid-responsive asthma / 천식및알레르기

BACKGROUND: Glucocorticoids (GC) are the most potent medications used to control airway inflammation associated with asthma. The aim of this study was to see whether alterations in GC receptor binding contribute to poor response to GC therapy in severe asthma. METHODS: Seventeen patients with severe persistent bronchial asthma were studied. Patients were classified as GC sensitive if their morning FEV1 increased >15% after a 1-week course of systemic GC (>prednisolone 40mg/day) and GC-resistant if they failed to increase >15%. GC receptor binding affinity for dexamethasone of peripheral blood mononuclear cells (PBMCs) were determined by using a radioligand binding assay and Scatchard analysis. RESULTS: There were no significant differences of age, serum IgE levels, peripheral blood eosinophils and atopic states between GC-resistant (n=10) and GC-responsive (n=7) groups. GC-resistant patients had significantly decreased GC receptor binding affinity (Kd=24.3+/-9.55) compared to the GC sensitive patients (Kd=13.5+/-1.48) and normal controls (n=6, Kd=4.24+/-1.09). CONCLUSION: These data suggest that decreased binding affinity of GC receptor is an important factor in clinical GC resistance in chronic severe asthma.