【摘要】： 我們最近的研究發(fā)現(xiàn),酒精抑制大鼠結(jié)腸運(yùn)動(dòng),該作用是由NO介導(dǎo)的,但其機(jī)制不清。已有報(bào)道,酒精可引起肝實(shí)質(zhì)細(xì)胞和枯否細(xì)胞中iNOS mRNA表達(dá)增高,Blanco等研究者也發(fā)現(xiàn),在人工培養(yǎng)的星形膠質(zhì)細(xì)胞中酒精刺激可通過(guò)NF-κB途徑使iNOS的基因表達(dá)上調(diào)。 核因子κB(nuclear factor-κB,NF-κB)作為轉(zhuǎn)錄因子,廣泛參與腫瘤、炎癥及免疫性疾病的發(fā)病過(guò)程,參與機(jī)體防御反應(yīng)、組織損傷和應(yīng)激、細(xì)胞分化和細(xì)胞凋亡以及腫瘤生長(zhǎng)抑制等過(guò)程的信息轉(zhuǎn)導(dǎo)。NF-κB包括NF-κB_1、NF-κB_2和某些癌基因蛋白(如RelA)等,通常與其抑制蛋白IκB結(jié)合,以二聚體的形式存在于胞漿中,當(dāng)細(xì)胞受到外界刺激時(shí),如病毒或細(xì)菌感染、紫外線照射、氧化應(yīng)激等,IκB被迅速磷酸化,釋放NF-κB使之轉(zhuǎn)入胞核內(nèi),結(jié)合于特定的κB序列,啟動(dòng)和調(diào)節(jié)眾多與免疫、炎癥反應(yīng)有關(guān)的基因轉(zhuǎn)錄。 我們推測(cè),酒精可能通過(guò)激活NF-κB、提高iNOS表達(dá)而引起了NO釋放。本課題主要是對(duì)該假設(shè)進(jìn)行驗(yàn)證。 材料與方法 實(shí)驗(yàn)選用健康雄性Wistar大鼠,實(shí)驗(yàn)前禁食18h�？焖贍奚笫�,制備近端結(jié)腸縱形肌肌條,在灌流肌槽中孵育并記錄肌條自發(fā)收縮活動(dòng)。以平滑肌肌條張力變化為指標(biāo),分別觀察經(jīng)PDTC(NF-κB的阻斷劑)和SMT(iNOS的阻斷劑)預(yù)處理后,酒精對(duì)結(jié)腸縱形肌肌條運(yùn)動(dòng)的影響,用Western blot技術(shù)測(cè)定胞漿中iNOS和IκB表達(dá)量,以及NF-κB在胞核中的表達(dá)量,免疫組織化學(xué)的方法定位結(jié)腸內(nèi)表達(dá)iNOS的細(xì)胞,NO試劑盒測(cè)定結(jié)腸組織NO釋放量。 所有數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)誤表示,采用單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)處理,以P＜0.05為顯著性差異界值。 實(shí)驗(yàn)結(jié)果: 1.酒精(8.7×10~(-4)M)明顯抑制離體結(jié)腸縱型肌肌條的自發(fā)收縮活動(dòng)。加入酒精后,記錄2～4分鐘,7～9分鐘,12～14分鐘和17～19分鐘,肌條張力的R值從1降低到0.90±0.02,0.87±0.02,0.89±0.03 and 0.87±0.01。 2.灌流液中分別加入PDTC(10~(-2)M)(NF-κB的阻斷劑)或SMT(10~(-3)M)(選擇性iNOS拮抗劑)后,再加入同樣劑量的酒精,酒精對(duì)結(jié)腸肌條運(yùn)動(dòng)的抑制作用明顯減弱。 3.酒精(0.17×10~(-3)M- 1.30×10~(-3)M)上調(diào)iNOS的表達(dá),其中以8.7×10~(-4)M的酒精作用最明顯。PDTC(10~(-2)M)明顯抑制酒精(8.7×10~(-4)M)對(duì)iNOS的表達(dá)的上調(diào)作用。 4.酒精(8.7×10~(-4)M)增加胞核中NF-κB的表達(dá)量,但減少胞漿中IκB的表達(dá)量。PDTC逆轉(zhuǎn)酒精的這種作用。 5.免疫組化發(fā)現(xiàn)iNOS在結(jié)腸肌間神經(jīng)叢中表達(dá)。 6.正常大鼠結(jié)腸中NO含量為1.112±0.128μmol/g,加入酒精后20分鐘,NO含量增加為7.194±0.497μmol/g(P＜0.05,n=6),PDTC預(yù)處理后再加入酒精,結(jié)腸中NO含量降為3.267±0.314μmol/g(P＜0.05,n=6) 結(jié)論: 酒精抑制結(jié)腸運(yùn)動(dòng)的作用是通過(guò)激活NF-κB,上調(diào)iNOS表達(dá),從而增加NO釋放量引起的。
[Abstract]:Our recent studies have found that alcohol inhibits colonic motility in rats, which is mediated by NO, but its mechanism is unclear. It has been reported that alcohol can increase the expression of iNOS mRNA in hepatic parenchyma cells and Kupffer cells. Blanco and other researchers have also found that alcohol stimulation can up-regulate the expression of iNOS gene through NF- 魏 B pathway in cultured astrocytes. Nuclear factor 魏 B (nuclear factor- kappa B (NF- 魏 B), as a transcription factor, is involved in the pathogenesis of tumor, inflammation and immune diseases, as well as in defense response, tissue damage and stress. NF-魏 B includes NF- kappa B _ 1, NF-魏 B _ 2 and some oncogene proteins (such as RelA), which usually bind to I 魏 B, an inhibitor of NF-魏 B, and some oncogene proteins such as NF-魏 B, NF-魏 B and some oncogene proteins, such as NF-魏 B, NF-魏 B and some oncogene proteins (such as RelA). In the form of dimer, when cells are stimulated by external stimuli, such as virus or bacterial infection, ultraviolet radiation, oxidative stress, etc., I kappa B is rapidly phosphorylated, releasing NF- 魏 B into the nucleus and binding to specific 魏 B sequences. Initiate and regulate the transcription of many genes associated with immune and inflammatory responses. We speculate that alcohol may cause NO release by activating NF- kappa B and increasing iNOS expression. The main purpose of this paper is to test the hypothesis. Materials and methods healthy male Wistar rats were selected and fasting for 18 hours before the experiment. The longitudinal muscle strips of proximal colon were prepared by rapid sacrifice of rats, and the spontaneous contraction activities of the strips were recorded and incubated in the perfusion groove. The effects of alcohol on the movement of longitudinal colonic muscle strips were observed after pretreatment with PDTC (NF- kappa B blocker) and SMT (iNOS (blocker of SMT (iNOS), and the expression of iNOS and IkB in cytoplasm were measured by Western blot technique. And the expression of NF- kappa B in nucleus, localization of iNOS-expressing cells in colon by immunohistochemical method, and determination of NO release in colon tissue by NO kit. All data were expressed by mean 鹵standard error and analyzed by one-way ANOVA (P < 0.05). Experimental results: 1. Alcohol (8.7 脳 10 ~ (- 4) M) significantly inhibited the spontaneous contractile activity of isolated longitudinally isolated colonic muscle strips. After alcohol was added, the R value of muscle tension decreased from 1 to 0.90 鹵0.02, 0.87 鹵0.02, 0.89 鹵0.03 and 0.87 鹵0.01. The muscle tension was recorded for 2 minutes, 7 minutes, 9 minutes, 12 minutes, 14 minutes and 17 minutes 19 minutes after alcohol was added, and the R value decreased from 1 to 0.90 鹵0.02, 0.87 鹵0.02, 0.89 鹵0.03 min. 2. When PDTC (10 ~ (- 2) M) (NF- kappa B blocker) or SMT (10 ~ (- 3) M) (selective iNOS antagonist) was added to the perfusate respectively, and the same dose of alcohol was added, the inhibitory effect of alcohol on the movement of colonic muscle strips was obviously weakened. 3. Alcohol (0.17 脳 10 ~ (- 3) M ~ (- 1.30) 脳 10 ~ (- 3) M) up-regulated the expression of iNOS. PDTC (10 ~ (- 2) M) significantly inhibited the up-regulation of iNOS expression by alcohol (8.7 脳 10 ~ (- 4) M). 4. Alcohol (8.7 脳 10 ~ (- 4) M) increased the expression of NF- 魏 B in nucleus, but decreased the expression of I 魏 B in cytoplasm. 5. Immunohistochemical study showed that iNOS was expressed in myenteric plexus of colon. 6. The content of NO in the colon of normal rats was 1.112 鹵0.128 渭 mol / g. 20 minutes after adding alcohol, the content of NO increased to 7.194 鹵0.497渭 mol / g (P < 0.05. After pretreatment with 6), PDTC, alcohol was added. The content of NO in colon decreased to 3.267 鹵0.314 渭 mol / g (P < 0.05 (n = 6) conclusion: alcohol can inhibit colonic motility by activating NF- kappa B and up-regulating the expression of iNOS, thus increasing the amount of NO release.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R363

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