【摘要】： 目的研究腫瘤壞死因子-α(TNF-α)在細(xì)菌脂多糖(LPS)下調(diào)小鼠胎肝細(xì)胞色素P450 3A(CYP3A)基因表達(dá)中的作用,并探討低劑量LPS預(yù)處理對CYP3A下調(diào)的抑制效應(yīng)。 方法本研究由5個(gè)實(shí)驗(yàn)組成:實(shí)驗(yàn)一、不給予妊娠小鼠任何處理,在妊娠第14、16、18 d剖殺孕鼠和出生后第4、21、70 d剖殺子鼠;實(shí)驗(yàn)二、經(jīng)腹腔注射給予妊娠第17 d小鼠LPS(500μg/kg),LPS處理后不同時(shí)點(diǎn)(2、12 h)剖殺孕鼠;實(shí)驗(yàn)三、經(jīng)腹腔注射給予妊娠第17 d小鼠不同劑量LPS(200、500μg/kg),LPS處理后12 h剖殺孕鼠;實(shí)驗(yàn)四、經(jīng)腹腔注射給予妊娠第17 d小鼠LPS(500μg/kg),部分動(dòng)物于LPS處理前30 min經(jīng)腹腔注射給予TNF-α合成抑制劑己酮可可堿(PTX,100 mg/kg)進(jìn)行預(yù)處理,LPS處理后12 h剖殺孕鼠;實(shí)驗(yàn)五、經(jīng)腹腔注射給予妊娠第17 d小鼠LPS(500μg/kg),部分動(dòng)物于LPS處理前24 h經(jīng)腹腔注射給予低劑量LPS(10μg/kg),高劑量組于LPS處理后1.5 h和12 h剖殺孕鼠。采用RT-PCR技術(shù)分析母鼠肝臟、胎盤和胎肝組織Cyp3a11、TNF-α和PXR mRNA表達(dá)水平,Western blot技術(shù)檢測胎肝組織CYP3A蛋白表達(dá)水平,ELISA法測定母鼠血清、羊水和胎肝組織TNF-α、IL-1β和IL-6蛋白含量。 結(jié)果妊娠第14 d后胎肝組織表達(dá)高水平的CYP3A,隨著胎兒發(fā)育CYP3A表達(dá)水平逐步增加,出生后第4d達(dá)到成年小鼠表達(dá)水平的80%。孕期母鼠接觸LPS顯著下調(diào)胎肝組織Cyp3a11 mRNA和CYP3A蛋白表達(dá)水平,并呈明顯的時(shí)間和劑量-效應(yīng)關(guān)系;PTX預(yù)處理顯著減弱LPS對胎肝CYP3A表達(dá)的下調(diào)。另一項(xiàng)研究結(jié)果顯示,LPS處理(500μg/kg)前30 min經(jīng)腹腔注射低劑量LPS(10μg/kg)顯著抑制高劑量LPS對母鼠血清、羊水和胎肝組織TNF-α的誘導(dǎo)作用;低劑量LPS預(yù)處理也顯著減弱LPS對母鼠肝臟和胎盤TNF-αmRNA的上調(diào)作用,而對胎肝TNF-αmRNA表達(dá)水平無顯著影響;與其對高劑量LPS誘導(dǎo)TNF-α表達(dá)的抑制效應(yīng)相一致,低劑量LPS預(yù)處理明顯減弱LPS對小鼠胎肝組織CYP3A表達(dá)的下調(diào)作用。 結(jié)論母鼠孕期接觸LPS顯著下調(diào)胎肝CYP3A表達(dá);源自母鼠血清和羊水的TNF-α至少部分參與了LPS對胎肝CYP3A的下調(diào)作用;低劑量LPS預(yù)處理通過抑制母鼠TNF-α的產(chǎn)生減弱LPS對胎肝CYP3A的下調(diào)作用。
[Abstract]:Objective To study the role of tumor necrosis factor-1 (TNF-1) in the down-regulation of the expression of cytochrome P450 3A (CYP3A) gene in mouse fetal hepatocyte cytochrome P450 3A (CYP3A), and to investigate the effect of low-dose LPS pretreatment on the down-regulation of CYP3A. Methods The study consisted of 5 experimental groups:1. No treatment was given to pregnant mice, and the rats were killed at the 14th, 16th and 18th day of gestation and the 4th, 21st and 70th day after birth. Kg), after LPS treatment, the pregnant rats were not killed at the same time (2,12 h); in the experiment, the rats were given different doses of LPS (200,500. mu.g/ kg) in the 17th day of gestation by intraperitoneal injection, and the pregnant rats were killed at 12 h after LPS treatment; and 4, LPS (500. mu.g/ kg) was given to the 17th day of pregnancy by intraperitoneal injection. Kg), in the first 30 minutes of LPS treatment, the mice were treated with the TNF-1 synthesis inhibitor hexanone (PTX,100 mg/ kg) for pretreatment, and the pregnant rats were killed at 12 h after LPS treatment; and 5, LPS (500. mu.g/ kg) in the 17th day of pregnancy was given by intraperitoneal injection. Kg), partial animals were given low dose of LPS (10. mu.g/ kg) by intraperitoneal injection 24 hours before LPS treatment and 1.5 h and 12 h after LPS treatment in high dose group. Methods: The expression of cyp3a11, TNF-1 and PXR mRNA in the liver, placenta and fetal liver tissues of female rats were analyzed by RT-PCR. The expression level of CYP3A protein in fetal liver was detected by Western blot, and the levels of TNF-1, IL-1 and IL-6 in the serum, amniotic fluid and fetal liver of the fetal liver were determined by Western blot. The results showed that the expression of CYP3A in the fetal liver after the 14th day of pregnancy increased gradually with the increase of the expression of CYP3A in the fetus, and the fourth day after birth reached the adult mouse. The levels of cyp3a11 mRNA and CYP3A protein in the fetal liver tissues were significantly reduced by exposure to LPS during pregnancy, and the time and dose-effect relationship was observed. The pretreatment of PTX significantly reduced LPS-to-fetal liver CYP. A further study showed that LPS treatment (500. mu.g/ kg) was injected intraperitoneally at a low dose of LPS (10. mu.g/ kg) to significantly inhibit the high dose of LPS in the serum, amniotic fluid and fetal liver tissue TNF of the maternal serum, amniotic fluid and fetal liver The effect of LPS on the expression of TNF-1 mRNA in fetal liver and placenta was not significantly affected by low-dose LPS pretreatment, and the expression of TNF-VEGF in fetal liver was not significantly affected by LPS pretreatment. The inhibitory effect was consistent, and the low-dose LPS pretreatment significantly reduced the LPS-induced CYP3A in the mouse fetal liver tissue. The effect of LPS on the regulation of CYP3A in the fetal liver was significantly reduced during the pregnancy. The low dose of LPS pretreatment was used to reduce the production of TNF-1 in the female mice and to decrease the impact of LPS on the fetal liver.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

【共引文獻(xiàn)】

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