【摘要】： 目的:膽囊收縮素(cholecystokinin,CCK)是一種重要的神經(jīng)調(diào)節(jié)肽。CCK在體內(nèi)存在多種活性片段如4肽、8肽、33肽、39肽和58肽等,其中八肽膽囊收縮素(cholecystokinin octapeptide,CCK-8)是其最主要的活性形式。CCK通過其受體發(fā)揮調(diào)節(jié)作用,CCK受體屬于G蛋白藕聯(lián)受體,根據(jù)其親和力以及生物學(xué)功能不同,CCK受體分為A、B兩種亞型。近年來研究顯示,CCK-8及其受體在哺乳類動(dòng)物體內(nèi)分布十分廣泛。已有文獻(xiàn)報(bào)道,CCK-8可緩解失血性休克,在內(nèi)毒素血癥患者的血液中CCK-8含量顯著升高。此后本室的系列研究證實(shí),內(nèi)、外源腦腸肽CCK-8確實(shí)具有抗內(nèi)毒素休克(endotoxin shock,ES)作用,可減輕ES發(fā)生早期的肺循環(huán)和體循環(huán)血流動(dòng)力學(xué)紊亂,其機(jī)制與調(diào)節(jié)氧化應(yīng)激有關(guān)。 在ES發(fā)病過程中,一方面脂多糖(lipopolysaccharide,LPS)所導(dǎo)致的氧化應(yīng)激,產(chǎn)生大量的超氧陰離子(O-·2);另一方面誘導(dǎo)包括血管平滑肌細(xì)胞(VSMC)在內(nèi)的機(jī)體多種細(xì)胞誘導(dǎo)型一氧化氮合酶(iNOS)表達(dá),產(chǎn)生大量的一氧化氮(NO),NO和O-·2形成過氧亞硝基陰離子(ONOO-)。NO、O-·2以及ONOO-參與啟動(dòng)機(jī)體氧化應(yīng)激、介導(dǎo)ES發(fā)病過程中肺循環(huán)和體循環(huán)血管反應(yīng)性異常改變,因此,O-·2的產(chǎn)生和清除機(jī)制研究是十分重要的。 超氧化物歧化酶(superoxide dismutase,SOD)是機(jī)體清除O-·2唯一的特異性酶,可使體內(nèi)的O-·2維持在一定水平。目前,在真核細(xì)胞中發(fā)現(xiàn)有3種SOD:分布于細(xì)胞漿的CuZn SOD,分布于線粒體的MnSOD和分泌到細(xì)胞外的EC SOD,其中MnSOD是一種誘生型酶,多種因素如氧化應(yīng)激、腫瘤壞死因子和LPS等,都可誘導(dǎo)MnSOD表達(dá)增加。 我們已往的研究結(jié)果顯示,VSMC存在CCK-8受體,CCK-8對(duì)LPS誘導(dǎo)的培養(yǎng)血管內(nèi)皮細(xì)胞SOD活性變化具有一定的調(diào)節(jié)效應(yīng),CCK-8作用的靶點(diǎn)可能是血管SOD和O-·2的生成系統(tǒng)。然而,CCK-8調(diào)節(jié)血管SOD活性變化的機(jī)制尚不清楚。由于MnSOD是誘生型酶且分布在與氧化應(yīng)激發(fā)生密切相關(guān)的線粒體,探索CCK-8對(duì)LPS誘導(dǎo)MnSOD基因表達(dá)的影響及其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制如受體機(jī)制是必要的。本實(shí)驗(yàn)擬探討CCK-8對(duì)LPS誘導(dǎo)血管平滑肌細(xì)胞MnSOD基因表達(dá)的影響,以闡明CCK-8抗ES作用的分子機(jī)制。 方法:選用雄性、健康Wistar大鼠(120±20g),無菌條件下分離大鼠胸主動(dòng)脈,用貼塊法培養(yǎng)大鼠胸主動(dòng)脈平滑肌細(xì)胞(TASMCs),傳代至對(duì)數(shù)生長期(實(shí)驗(yàn)用5~8代),調(diào)整細(xì)胞密度為每瓶1×107,加入無血清DMEM培養(yǎng)液以及不同處理因素,檢測(cè)MnSOD mRNA的表達(dá)情況。 實(shí)驗(yàn)程序:1、分別用0.01mg/L、0.1mg/L、1mg/L LPS以及生理鹽水孵育TASMCs 4h,用0.1mg/L LPS孵育TASMCs 2h、4h、8h,檢測(cè)MnSOD mRNA表達(dá)變化,以觀察LPS誘導(dǎo)TASMCs MnSOD mRNA表達(dá)的時(shí)效與量效關(guān)系。2、用0.1mg/L LPS分別和10-6、10-8、10-10mol/L CCK-8共同處理細(xì)胞4h,檢測(cè)MnSOD mRNA表達(dá)變化,以觀察CCK-8對(duì)LPS誘導(dǎo)TASMCs MnSOD mRNA表達(dá)的影響。3、在前述實(shí)驗(yàn)結(jié)果的基礎(chǔ)上,選擇CCK-8、LPS各一個(gè)劑量和一個(gè)時(shí)間點(diǎn),CCK-8+LPS與丙谷胺(Pro,CCK受體非特異性阻斷劑)、CR-1409(CCK-A受體特異性阻斷劑)或CR-2945(CCK-B受體特異性阻斷劑)處理細(xì)胞,另設(shè)Pro、CR-1409、CR-2945對(duì)照組,檢測(cè)MnSOD mRNA表達(dá)變化,研究CCK-8對(duì)LPS誘導(dǎo)TASMCs MnSOD mRNA表達(dá)影響的受體機(jī)制。 用RT-PCR方法檢測(cè)MnSOD基因表達(dá),用江蘇捷達(dá)凝膠分析軟件對(duì)電泳譜帶進(jìn)行半定量分析,用MnSOD與β-actin的吸光度比值代表MnSOD mRNA相對(duì)表達(dá)水平。數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,用SPSS13.0統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,組間比較行單因素方差分析(one-way ANOVA),有顯著差異者進(jìn)一步用最小顯著差法(LSD)進(jìn)行兩兩比較,以P0.05為有統(tǒng)計(jì)學(xué)意義。 結(jié)果:1、在TASMCs存在MnSOD mRNA基礎(chǔ)表達(dá);與對(duì)照組相比,0.01mg/L、0.1mg/L及1.0mg/L LPS分別孵育細(xì)胞4h可劑量依賴性引起MnSOD mRNA表達(dá)升高(P0.05);在LPS誘導(dǎo)TASMCs MnSOD表達(dá)的時(shí)效關(guān)系中,發(fā)現(xiàn)2h就可誘導(dǎo)MnSOD mRNA表達(dá)升高(P0.05),4h表達(dá)達(dá)到高峰(P0.05),8h持續(xù)高表達(dá)(P0.05)。2、10-6、10-8、10-10mol/L CCK-8預(yù)先處理TASMCs 30min后加入LPS,MnSOD mRNA表達(dá)可不同程度地被抑制,與LPS組相比有顯著性差異(P0.05);CCK-8的抑制效應(yīng)具有濃度依賴性(P0.05);CCK-8(10-8 mol/L)單獨(dú)處理細(xì)胞可導(dǎo)致TASMCs MnSOD mRNA表達(dá)明顯下調(diào),與對(duì)照組相比有顯著性差異(P0.05)。3、預(yù)先用CR-1409、CR-2945、Pro孵育細(xì)胞10min后再給予CCK-8和LPS,CR-1409+CCK-8+LPS和CR-2945+CCK-8+LPS組MnSOD mRNA表達(dá)與CCK-8+LPS組相比明顯升高(P0.05),但仍低于LPS組(P0.05),其中CR-2945的拮抗作用比CR-1409更為顯著(P0.05);Pro+CCK-8+LPS組則完全翻轉(zhuǎn)了CCK-8的抑制效應(yīng)。 結(jié)論:CCK-8對(duì)TASMCs MnSOD mRNA的基礎(chǔ)表達(dá)具有抑制性調(diào)節(jié)作用;LPS可劑量依賴性誘導(dǎo)TASMCs MnSOD mRNA表達(dá)上調(diào),MnSOD mRNA的表達(dá)高峰開始于LPS作用后4h左右;CCK-8可濃度依賴性抑制LPS誘導(dǎo)TASMCs MnSOD mRNA表達(dá),CCK受體介導(dǎo)CCK-8該抑制作用,其中CCK-BR可能起主要作用。
[Abstract]:Objective: The cholecystokinin (CCK) is an important neuroregulatory peptide. The CCK-8 is the most active form of CCK-8 in the presence of a variety of active fragments such as 4-peptide,8-peptide,33-peptide,39-peptide, and 58-peptide. CCK receptor plays a regulating role through its receptor, and the CCK receptor belongs to the G-protein lotus-linked receptor, and according to its affinity and biological function, the CCK receptor is divided into two subtypes A and B. In recent years, it is shown that CCK-8 and its receptor are widely distributed in mammals. It has been reported that CCK-8 can alleviate the hemorrhagic shock, and the content of CCK-8 in the blood of patients with endotoxemia is significantly increased. The series of studies in this room confirmed that the exogenous brain-intestinal peptide (CCK-8) did have an anti-endotoxin shock (ES) function, which can reduce the early lung circulation and the circulatory disturbance of the body circulation in the ES, and the mechanism is related to the regulation of oxidative stress. In the pathogenesis of ES, the oxidative stress induced by lipopolysaccharides (LPS), on the one hand, produces a large amount of superoxide anion (O-2), and on the other hand, induces a variety of cell-inducible nitric oxide synthase (i), including vascular smooth muscle cells (VSMC), Nitric oxide (NO), O-路 2 and ONOO-2 form a peroxynitrite anion (ONOO-). NO, O-路 2 and ONOO-participate in the initiation of oxidative stress in the body. A Study on the Mechanism of the Generation and Removal of O-路 2 It is very important. Superoxide dismutase (SOD) is the only specific enzyme for the body to remove O-路 2, which can make the body At present, three kinds of SOD are found in the eukaryotic cells: CuZn SOD distributed in the cytoplasm, MnSOD distributed in the mitochondria and EC SOD secreted outside the cell, wherein the MnSOD is a kind of induced type enzyme, various factors such as oxidative stress, tumor necrosis factor and LPS, etc., The results showed that the expression of CCK-8 and CCK-8 in VSMC had some effect on the changes of SOD activity in cultured vascular endothelial cells induced by LPS and CCK-8. The target point may be the production system of blood vessel SOD and O-路 2. However, CCK -8. The mechanism of regulating the activity of SOD in blood vessel is not clear. Because MnSOD is an induced type enzyme and is distributed in the mitochondria closely related to oxidative stress, the expression of CCK-8 on the induction of MnSOD gene induced by LPS is explored. The effects of CCK-8 on the expression of MnSOD gene in vascular smooth muscle cells induced by LPS were discussed in this experiment. In order to elucidate the molecular mechanism of the anti-ES effect of CCK-8, male and healthy Wistar rats (120-20 g) were used to separate the thoracic aorta from the rat thoracic aorta under aseptic conditions, and the rat thoracic aortic smooth muscle cells (TASMCs) were cultured by means of the patch method. The growth phase (5-8 for the experiment) was adjusted, the cell density was adjusted to 1-107 per bottle, and the serum-free DMEM culture was added. The expression of MnSOD mRNA was detected by incubation of TASMCs at 0.01 mg/ L, 0.1 mg/ L,1 mg/ L LPS and physiological saline respectively. And 10-6,10-8,10-10 mol/ L CCK-8 co-treated the cells for 4 hours to detect the change of the expression of MnSOD mRNA in order to observe the effect of CCK-8 on the expression of TASMCs MnSOD mRNA induced by LPS. The expression of MnSOD mRNA was detected by the control group of Pro, CR-1409 and CR-2945, and the expression of MnSOD mRNA was detected by the control group of Pro, CR-1409, and CR-2945, and the expression of MnSOD mRNA was detected by the control group of Pro, CR-1409 and CR-2945. The receptor mechanism of the effect of CK-8 on the expression of TASMCs MnSOD mRNA was induced by LPS. The expression of MnSOD gene was detected by RT-PCR. The relative expression level of MnSOD mRNA was represented by the ratio of the absorbance of MnSOD and P-actin to the level of the relative expression of MnSOD mRNA. The data was expressed by the standard deviation (x% s) of the mean number of MnSOD, and the statistical analysis was carried out by using the SPSS13.0 statistical analysis software, and the one-way ANOVA was compared among the groups. The results were as follows:1. The expression of MnSOD mRNA was in the presence of TASMCs. Compared with the control group, the dose-dependent manner of the cells were respectively incubated with 0.01 mg/ L, 0.1 mg/ L and 1.0 mg/ L of LPS. The expression of MnSOD mRNA was increased (P0.05). In the time-aging relationship of the expression of TASMCs MnSOD induced by LPS, it was found that the expression of MnSOD mRNA was increased (P0.05). The expression of MnSOD mRNA was high (P0.05). The inhibitory effect of CCK-8 (10-8mol/ L) on the expression of TASMCs MnSOD was significantly lower than that of the control group (P <0.05), and the expression of CCK-8 (10-8 mol/ L) alone could lead to a significant decrease in the expression of TASMCs MnSOD mRNA, and the expression of CCK-8 and LPS, CR-1409 + CCK-8 + LPS and CR-2945 + CCK-8 + LPS group MnS were given in advance after 10 min incubation with CR-1409, CR-2945, and Pro. The expression of OD mRNA was significantly higher than that of the CCK-8 + LPS group (P0.05), but still lower than that of the LPS group (P0.05), in which the antagonistic effect of CR-2945 was higher than that of the group C. The results showed that CCK-8 had an inhibitory effect on the basal expression of TASMCs MnSOD mRNA. Right; CCK-8 concentration-dependent inhibition of L
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：D919

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本文編號(hào)：2500759