内皮型一氧化氮合酶基因缺失小鼠的脑细胞色素P450-1B1蛋白表达及其意义

Expression and role of cytochrome P450-1B1 in the brains of endothelial nitric oxide synthase gene knockout mice

目的研究内皮型一氧化氮合酶(e NOS)基因敲除小鼠脑内细胞色素P450-1B1(CYP1B1)蛋白的表达变化及其意义。方法按照体重将小鼠分为4组:野生型组(n=4)、e NOS基因敲除组(n=4)、实验组(2种小鼠各4只)和对照组(2种小鼠各4只)。实验组腹腔注射2,3',4,5'-tetramethoxystilbene(TMS,CYP1B1抑制剂) 300μg·kg^(-1),对照组腹腔注射等剂量溶剂二甲基亚砜30μL,每天1次,连续1周。以免疫荧光染色和免疫印迹法测定小鼠的CYP1B1与半胱氨酸天冬氨酸蛋白酶-3(Caspase-3) p17表达。结果野生型组和基因敲除组小鼠的前脑皮质区CYP1B1免疫荧光阳性细胞数分别是(106±21)个/200×视野、(249±17)个/200×视野;这2组在纹状体区阳性细胞数分别是(85±16)个/200×、(211±23)个/200×视野,2组比较差异均有统计学意义(均P <0. 001)。野生型组和基因敲除组小鼠的脑CYP1B1蛋白表达灰度值分别是0. 45±0. 04,1. 15±0. 15,2组比较差异均有统计学意义(均P <0. 001)。TMS干预后,实验组中野生型小鼠和e NOS基因敲除小鼠脑Caspase-3 p17蛋白灰度值分别是1. 24±0. 21,2. 21±0. 17,均显著高于对照组中的野生型小鼠和e NOS基因敲除小鼠,Caspase-3 p17蛋白灰度值分别是0. 23±0. 03,0. 76±0. 08,组间比较差异均有统计学意义(均P <0. 001)。结论 e NOS基因敲除小鼠脑内CYP1B1蛋白表达升高并且起到抗凋亡作用,但其作用机制目前尚不明确。

Objective To investigate the expression and role of cytochrome P450-1 B1( CYP1 B1) in endothelial nitric oxide synthase( eNOS) gene knockout mice brains. Methods The animals were divided into 4 groups: wild type group( n = 4),eNOS gene knockout group( n = 4),experimental group( 4 mouse of two species) and control group( 4 mouse of two species). The experimental group and control group was intraperitoneal injection with CYP1 B1 inhibitor 2,3’,4,5’-tetramethoxystilbene( TMS,300 μg ·kg-1) and isodose solvent dimethylsulfoxide( DMSO,30 μL) respectively,once a day for 1 week. The protein expressions of CYP1 B1 and Caspase-3 p17 were determined by immunofluorescence staining and Western blot. Results The number of CYP1 B1 immunofluorescence positive cells in the frontal cortex in the wild type group,eNOS gene knockout group were 106 ± 21/200 × fields,249 ± 17/200 × fields;the No. of the cells in the striatum of the two groups were 85 ± 16/200 × fields,211 ± 23/200 × fields. Comparison between wild type group with eNOS gene knockout group,the difference was significantly( all P < 0. 01). The gray level of CYP1 B1 protein expression in the wild-type group and knockout group was 0. 45 ± 0. 04 and 1. 15 ± 0. 15,respectively. The difference between the two groups was significantly( P < 0. 001). After TMS intervention,the expression level of caspase-3 p17 protein in the brain of wild-type mice and e NOS knockout mice in the experimental group was significantly increased,the gray value of caspase-3 p17 protein was 1. 24 ± 0. 21,2. 21 ± 0. 17,both significantly higher than the corresponding control group,the gray value of caspase-3 p17 protein was 0. 23 ± 0. 03,0. 76 ± 0. 08,respectively. The differences were statistically significant( all P < 0. 001). Conclusion The expression of CYP1 B1 in the brains of eNOS knockout mice is increased and has anti-apoptotic effect,but the mechanism underlie this remains unclear.