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Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells
Journal of ZheJiang University(Medical Science), 2013, 42(1): 38-44.
https://doi.org/10.3785/j.issn.1008-9292.2013.01.007
Objective: To investigate the optimal conditions of tri-expression of CYP3A4,POR and cyt b5 in Sf 9 cells. Methods: The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks.The optimized conditions,including the temperature and rotation speed,the culture volume,the amount of surfactant and the culture time were studied.The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. Results: When the temperature and rotation speed of the shaker were 27℃ and 90 r/min,the cell density and culture volume were 5×105 cells/ml and 80-120 ml per 250 ml shaker flasks,respectively.When Pluronic F-68 was 0.1% and the culture time was 72 h,the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins.When the ratio of the volume of three added viruses was 1∶1∶1,the expression condition was optimal,under which the Km,Vmax,and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min·g protein)and 4.34 ml/(min·g protein),respectively. Conclusions: The conditions of tri-expressing of CYP3A4,POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.
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Effects of spironolactone on type Ⅰ,Ⅲ collagen concentration in myocardium of spontaneous hypertension rats
Journal of ZheJiang University(Medical Science), 2013, 42(1): 81-85.
https://doi.org/10.3785/j.issn.1008-9292.2013.01.013
Objective: To investigate the effects of spironolactone on the concentration of collagen type Ⅰ,Ⅲ in the myocardium of spontaneous hypertension rats (SHR). Methods: Twenty 8-week male SHR were assigned randomly into spironolactone (SHR-SPIRO,n=10) and control groups (SHR-CON,n=10),sex-age matched Wistar Kyoto rats (WKY group,n=7) were also served as controls.The rats of SHR-SPIRO group were given 20 mg/(kg·d) of spironolactone,the rats of SHR-CON and WKY groups were given the same volume of distilled water.After 16 weeks,the concentration of collagen type I was analyzed with Western blot.The areas of collagen typeⅠand Ⅲ were observed under polarized light microscopy and the ratio of typeⅠ/Ⅲ collegen was calculated through accumulation score. Results: Compared with WKY group,the concentration of collagen type Ⅰ in SHR-CON group was significantly higher (1.87±0.2 vs 1.21±0.7,P<0.05).After 16 weeks of treatment the concentration of collagen type Ⅰ(1.42±0.05 vs 1.87±0.2,P<0.05) and Ⅰ/Ⅲ ratio in SHR-SPIRO group were significantly reduced (15.64±1.34 vs 20.8±3.04,P<0.05) compared with SHR-CON group; but there were no differences in accumulation area scores of collagen type Ⅲ among three groups(368.3±30.2 vs 481.6±32.4 vs 406.2±45.3,P>0.05). Conclusions: The deposition of collagen type Ⅰ in myocardium may be involved in myocardial fibrosis of SHR,and spironolactone can decrease the concentration of collagen type I,which may be one of the mechanisms for its therapeutic effects.
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19 articles
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