CCK-8对LPS诱导大鼠胸主动脉血管平滑肌细胞MnSOD基因表达的影响
发布时间：2019-06-16 19:06
【摘要】： 目的:胆囊收缩素(cholecystokinin,CCK)是一种重要的神经调节肽。CCK在体内存在多种活性片段如4肽、8肽、33肽、39肽和58肽等,其中八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)是其最主要的活性形式。CCK通过其受体发挥调节作用,CCK受体属于G蛋白藕联受体,根据其亲和力以及生物学功能不同,CCK受体分为A、B两种亚型。近年来研究显示,CCK-8及其受体在哺乳类动物体内分布十分广泛。已有文献报道,CCK-8可缓解失血性休克,在内毒素血症患者的血液中CCK-8含量显著升高。此后本室的系列研究证实,内、外源脑肠肽CCK-8确实具有抗内毒素休克(endotoxin shock,ES)作用,可减轻ES发生早期的肺循环和体循环血流动力学紊乱,其机制与调节氧化应激有关。 在ES发病过程中,一方面脂多糖(lipopolysaccharide,LPS)所导致的氧化应激,产生大量的超氧阴离子(O-·2);另一方面诱导包括血管平滑肌细胞(VSMC)在内的机体多种细胞诱导型一氧化氮合酶(iNOS)表达,产生大量的一氧化氮(NO),NO和O-·2形成过氧亚硝基阴离子(ONOO-)。NO、O-·2以及ONOO-参与启动机体氧化应激、介导ES发病过程中肺循环和体循环血管反应性异常改变,因此,O-·2的产生和清除机制研究是十分重要的。 超氧化物歧化酶(superoxide dismutase,SOD)是机体清除O-·2唯一的特异性酶,可使体内的O-·2维持在一定水平。目前,在真核细胞中发现有3种SOD:分布于细胞浆的CuZn SOD,分布于线粒体的MnSOD和分泌到细胞外的EC SOD,其中MnSOD是一种诱生型酶,多种因素如氧化应激、肿瘤坏死因子和LPS等,都可诱导MnSOD表达增加。 我们已往的研究结果显示,VSMC存在CCK-8受体,CCK-8对LPS诱导的培养血管内皮细胞SOD活性变化具有一定的调节效应,CCK-8作用的靶点可能是血管SOD和O-·2的生成系统。然而,CCK-8调节血管SOD活性变化的机制尚不清楚。由于MnSOD是诱生型酶且分布在与氧化应激发生密切相关的线粒体,探索CCK-8对LPS诱导MnSOD基因表达的影响及其信号转导机制如受体机制是必要的。本实验拟探讨CCK-8对LPS诱导血管平滑肌细胞MnSOD基因表达的影响,以阐明CCK-8抗ES作用的分子机制。 方法:选用雄性、健康Wistar大鼠(120±20g),无菌条件下分离大鼠胸主动脉,用贴块法培养大鼠胸主动脉平滑肌细胞(TASMCs),传代至对数生长期(实验用5~8代),调整细胞密度为每瓶1×107,加入无血清DMEM培养液以及不同处理因素,检测MnSOD mRNA的表达情况。 实验程序:1、分别用0.01mg/L、0.1mg/L、1mg/L LPS以及生理盐水孵育TASMCs 4h,用0.1mg/L LPS孵育TASMCs 2h、4h、8h,检测MnSOD mRNA表达变化,以观察LPS诱导TASMCs MnSOD mRNA表达的时效与量效关系。2、用0.1mg/L LPS分别和10-6、10-8、10-10mol/L CCK-8共同处理细胞4h,检测MnSOD mRNA表达变化,以观察CCK-8对LPS诱导TASMCs MnSOD mRNA表达的影响。3、在前述实验结果的基础上,选择CCK-8、LPS各一个剂量和一个时间点,CCK-8+LPS与丙谷胺(Pro,CCK受体非特异性阻断剂)、CR-1409(CCK-A受体特异性阻断剂)或CR-2945(CCK-B受体特异性阻断剂)处理细胞,另设Pro、CR-1409、CR-2945对照组,检测MnSOD mRNA表达变化,研究CCK-8对LPS诱导TASMCs MnSOD mRNA表达影响的受体机制。 用RT-PCR方法检测MnSOD基因表达,用江苏捷达凝胶分析软件对电泳谱带进行半定量分析,用MnSOD与β-actin的吸光度比值代表MnSOD mRNA相对表达水平。数据用均数±标准差(x±s)表示,用SPSS13.0统计分析软件进行统计学分析,组间比较行单因素方差分析(one-way ANOVA),有显著差异者进一步用最小显著差法(LSD)进行两两比较,以P0.05为有统计学意义。 结果:1、在TASMCs存在MnSOD mRNA基础表达;与对照组相比,0.01mg/L、0.1mg/L及1.0mg/L LPS分别孵育细胞4h可剂量依赖性引起MnSOD mRNA表达升高(P0.05);在LPS诱导TASMCs MnSOD表达的时效关系中,发现2h就可诱导MnSOD mRNA表达升高(P0.05),4h表达达到高峰(P0.05),8h持续高表达(P0.05)。2、10-6、10-8、10-10mol/L CCK-8预先处理TASMCs 30min后加入LPS,MnSOD mRNA表达可不同程度地被抑制,与LPS组相比有显著性差异(P0.05);CCK-8的抑制效应具有浓度依赖性(P0.05);CCK-8(10-8 mol/L)单独处理细胞可导致TASMCs MnSOD mRNA表达明显下调,与对照组相比有显著性差异(P0.05)。3、预先用CR-1409、CR-2945、Pro孵育细胞10min后再给予CCK-8和LPS,CR-1409+CCK-8+LPS和CR-2945+CCK-8+LPS组MnSOD mRNA表达与CCK-8+LPS组相比明显升高(P0.05),但仍低于LPS组(P0.05),其中CR-2945的拮抗作用比CR-1409更为显著(P0.05);Pro+CCK-8+LPS组则完全翻转了CCK-8的抑制效应。 结论:CCK-8对TASMCs MnSOD mRNA的基础表达具有抑制性调节作用;LPS可剂量依赖性诱导TASMCs MnSOD mRNA表达上调,MnSOD mRNA的表达高峰开始于LPS作用后4h左右;CCK-8可浓度依赖性抑制LPS诱导TASMCs MnSOD mRNA表达,CCK受体介导CCK-8该抑制作用,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
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919
[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
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919
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