细胞色素P450 CYP4A的组织和细胞表达特异性  

Specificity of cytochrome P-450 CYP4A expression in tissues and cells

作　　者：陆刚 唐晓波[2] 鲍岩岩[2] 余辉艳[2] 郑蕾[3] 朱大岭[2] 

机构地区：[1]哈药集团生物工程有限公司,黑龙江哈尔滨150086 [2]哈尔滨医科大学药学院,黑龙江哈尔滨150081 [3]哈尔滨医科大学附属第一临床医学院,黑龙江哈尔滨150081

出　　处：《中国药理学与毒理学杂志》2007年第6期505-511,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目(30370578);国家自然科学基金资助项目(30470752)~~

摘　　要：目的确定细胞色素P450 CYP4A在组织和细胞水平表达是否具有特异性。方法采用Western印迹法分析细胞色素P450 CYP4A蛋白在培养的牛肺动脉内皮细胞、平滑肌细胞及牛肾动脉内皮细胞的表达;采用原位杂交法检测细胞色素P450 CYP4A mRNA在大鼠肺支气管内皮和血管内皮细胞的表达;以14C标记的花生四烯酸(AA)作为反应底物,用荧光高效液相色谱法(HPLC)通过检测牛肺动脉内皮细胞催化外源性AA生成20-羟基廿碳四烯酸(20-HETE)的反应,确定肺动脉内皮细胞是否存在细胞色素P450 CYP4A;将肺动脉内皮细胞破碎并用乙酸乙酯萃取,用荧光物质标记萃取物,用荧光HPLC检测内源性20-HETE,从代谢产物水平确定细胞色素P450 CYP4A的功能蛋白质表达。结果①Western印迹法分析结果表明,细胞色素P450 CYP4A蛋白在牛肺动脉内皮细胞和平滑肌细胞中均有表达,在肺动脉内皮细胞中的表达明显高于平滑肌细胞(积分吸光度值分别为10182±279,5249±167);在牛肾动脉内皮细胞中也有表达,但明显低于肺动脉内皮细胞的表达(积分吸光度值分别为12173±171,17863±207)。②原位杂交结果显示,细胞色素P450 CYP4A mRNA可在大鼠肺支气管内皮细胞和大鼠肺血管内皮均有表达。③用HPLC在牛肺动脉内皮细胞中检测到20-HETE,表明细胞色素P450 CYP4A在肺动脉内皮细胞存在。结论细胞色素P450 CYP4A有其特定的组织和细胞分布,在牛和大鼠肺脏动脉内皮细胞有高表达。AIM Ascertaining whether or not having specific expression of cytochrome P-450 CYP4A on the tissular and cellular level. METHODS The expression of cytochrome P- 450 CYP4A protein was detected by Western blot in cultured bovine pulmonary and kidney arterial endothelia as well as in pulmonary artery smooth muscle cells. The expression of cytochrome P-450 CYP4A mRNA was detected by in situ hybridization in bronchus endothelia and vascular endothelial cells of lung tissue slice. The location of cytochrome P-450 CYP4A enzyme in pulmonary artery endothelial cells was determined by constructing warm incubate system, the reaction substrate was ^14C labeled arachidonic acid （AA）, and by labeling cell extraction with fluorescent material, 20-HETE was detected by high efficiency liquid chromatography. RESULTS ① Western blot indicated cytochrome P-450 CYP4A protein expressed in bovine pulmonary arterial endothelia cells as well as in pulmonary artery smooth muscle cells, and more expressed in endothelia cells （integrated opitical absorbance 10 182 ± 279,5249±167）, cytochrome P-450 CYP4A pro-ein expressed also in bovine kidney arterial en-othelial cells, and more expressed in lungs （integrated opitical absorbance 12 173 ±171, 17 863± 207 ）. ② In situ hybridization displayed cytochrome P-450 CYP4A mRNA expressed not only in rat lung bronchus endothelial cell but in rat lung vascular endothelial cells. ③20-HETE was detected by fluorescence HPLC in bovine pulmonary artery endothelial cells. CONCLUSION Cytochrome P- 450 CYP4A has definite histiocyte distribution, and expresses more in bovine and rat pulmonary artery endothelial cells.

关 键 词：细胞色素P450 CYP4A 细胞 培养的 蛋白表达 基因表达 组织分布 

分 类 号：R341[医药卫生—基础医学]