CUEDC2对急性胰腺炎腺泡细胞TNF- α、IL-6的影响

发布时间：2018-05-14 07:32

本文选题：急性胰腺炎 + CUEDC2　；参考：《河北医科大学》2017年硕士论文

【摘要】：急性胰腺炎(acutepancreatitis,AP)是临床常见急腹症,具有起病急、病情重、变化快等特点,除了胰腺本身病变之外,还可能引起胰腺外器官和组织的损伤,甚至最终诱发全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)、多器官功能障碍综合征(multiple-organ dysfunction syndrome,MODS)等而导致死亡。尽管在过去的几十年里急性胰腺炎的研究取得明显进展,但是其发病机制仍较复杂。急性胰腺炎(acute pancreatitis,AP)无论病因如何,损伤的胰腺会引起胰酶的自身消化,同时作为炎症刺激物刺激单核巨噬细胞及胰腺腺泡细胞释放炎症介质和细胞因子,触发炎症介质形成瀑布样级联反应,最终会导致局部或全身炎症反应。CUEDC2(CUE domain-containing 2)是一种含有CUE结构域的多功能蛋白质,广泛在人类组织和器官中表达。大量研究表明,CUEDC2可以作用于多种细胞内信号转导通路,参与许多细胞活动,如细胞周期的调节,生长信号因子的转导、肿瘤的发生发展及炎症反应。目前研究发现CUEDC2不仅与肿瘤的发生发展密切相关,如乳腺癌、结肠炎相关性肿瘤、肺癌等,还可以参与炎症反应,如结肠炎等。关于CUEDC2与急性胰腺炎的炎症反应中的关系尚未有研究报道。本研究拟初步探讨CUEDC2在急性胰腺炎中的作用。目的:本研究通过雨蛙素诱导AR42J细胞建立急性胰腺炎细胞模型,并利用质粒转染技术过表达CUEDC2。监测细胞培养液中淀粉酶(amylase,AMY)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)的水平,初步探讨CUEDC2对急性胰腺炎腺泡细胞TNF-α、IL-6的影响。方法:1应用雨蛙素诱导AR42J细胞建立急性胰腺炎细胞模型。2获得过表达CUEDC2的AR42J细胞:采用脂质体转染法将CUEDC2质粒转入AR42J细胞中,应用荧光显微镜查看转染效率。3实验分组:AR42J细胞分为4组:阴性质粒对照组(Neg-Control组)、阴性质粒雨蛙素组(Neg-CAE组)、CUEDC2质粒对照组(CUEDC2-Control组)、CUEDC2质粒雨蛙素组(CUEDC2-CAE组)。具体分组方法如下:阴性质粒组:应用阴性质粒转染AR42J细胞。Neg-CAE组给予10-7 mol/L雨蛙素,Neg-Control组给予等体积完全培养基,再将两组分为3个亚组:4h、8h、24h。CUEDC2质粒组:应用CUEDC2质粒转染AR42J细胞。CUEDC2-CAE组给予10-7mol/L雨蛙素 CUEDC2-Control组给予等体积完全培养基,再将两组分为3个亚组:4h、8 h、24h。4 CUEDC2 测定:PCR 检测 CUEDC2 mRNA 的表达。western-blot检测CUEDC2蛋白的表达。5淀粉酶、TNF-α、IL-6测定:应用淀粉酶试剂盒检测细胞上清中淀粉酶(amylase,AMY)水平,应用 ELISA(enzyme linked immunosorbent assay)方法检测细胞培养上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及白细胞介素-6(interleukin-6,IL-6)水平。6统计学方法:应用SPSS 21.0软件包进行统计学处理,P0.05认为具有统计学意义。结果:1成功建立了急性胰腺炎细胞模型,雨蛙素组AMY的水平随时间延长而增高且均较对照组升高(P0.05)。雨蛙素组中CUEDC2mRNA水平较对照组下降(P0.05)。雨蛙素组中各个时间点的CUEDC2mRNA无明显变化(P0.05);2应用CUEDC2质粒转染细胞后,可见绿色荧光颗粒,细胞生长状态良好,表明转染条件稳定,细胞转染成功,转染率约为65%-70%。CUEDC2质粒组CUEDC2蛋白水平较对照组升高(P0.05)。阴性质粒组与对照组相比未见明显差异(P0.05);3细胞培养上清AMY:各个时间点比较,在阴性质粒对照组和CUEDC2质粒对照组中AMY水平未见明显上升,且两组之间未见明显差异(P0.05);在各个时间点AMY水平,阴性质粒对照组和CUEDC2质粒对照之间无显著差异(P0.05)。在阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组中AMY水平随时间逐渐增高(P0.05)。阴性质粒雨蛙素组与CUEDC2质粒雨蛙素组均较各自对照组升高(P0.05);CUEDC2质粒雨蛙素组与阴性质粒雨蛙素组相比AMY水平下降(P0.05);4细胞培养上清TNF-α、IL-6:在阴性质粒对照组和CUEDC2质粒对照组中各个时间点TNF-α、IL-6水平较低,且两组之间无统计学意义(P0.05);在阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组中随时间的延长TNF-α、IL-6水平逐渐升高(P0.05)。阴性质粒雨蛙素组与CUEDC2质粒雨蛙素组在各个时间点TNF-α、IL-6水平均较各自对照组显著上升(P0.05);CUEDC质粒雨蛙素组与阴性质粒雨蛙素组相比,在各个时间点TNF-α、IL-6水平均降低(P0.05)。结论:1应用雨蛙素诱导可成功制备AP细胞模型。2在AP细胞模型中CUEDC2 mRNA的水平下调。3成功构建了过表达CUEDC2蛋白的AP细胞模型,为研究CEUDC2在急性胰腺炎中的作用奠定了基础。4过表达CUEDC2后,雨蛙素诱导的AR42J细胞产生AMY、TNF-α及IL-6水平降低,说明CUEDC2能抑制炎性介质的过度增长,在急性胰腺炎中其保护作用。
[Abstract]:Acutepancreatitis (AP) is a common clinical acute abdomen, which has the characteristics of acute onset, severe disease and rapid change. In addition to the pancreatic disease, it may also cause injury to the external organs and tissues of the pancreas, and even eventually induce systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), multiple organ dysfunction. Death is caused by multiple-organ dysfunction syndrome (MODS). Although significant progress has been made in the study of acute pancreatitis in the past few decades, its pathogenesis is still more complex. Acute pancreatitis (acute pancreatitis, AP), regardless of the cause of the disease, causes the pancreas to cause the digestion of the pancreatin, as well as the inflammation of the pancreas. The irritant stimulates mononuclear macrophages and pancreatic acinar cells to release inflammatory mediators and cytokines, triggering the inflammatory mediators to form a cascade cascade, which eventually leads to local or systemic inflammatory response.CUEDC2 (CUE domain-containing 2), a multipotent protein containing the CUE domain, which is widely expressed in human tissues and organs. A large number of studies have shown that CUEDC2 can play a role in a variety of intracellular signal transduction pathways involved in many cell activities, such as cell cycle regulation, growth signal transduction, tumor development, and inflammatory reactions. Current studies have found that CUEDC2 is not only closely related to the development of tumors, such as breast cancer, colitis associated tumor, lung Cancer and so on, can also participate in inflammatory reactions, such as colitis, etc. the relationship between CUEDC2 and acute pancreatitis has not been reported. This study intends to preliminarily explore the role of CUEDC2 in acute pancreatitis. Objective: to induce AR42J cells to establish acute pancreatitis cell model by using rain frog hormone and to use plasmid transfection technique. The effects of CUEDC2. on the level of amylase, AMY, tumor necrosis factor- alpha, TNF- alpha, and interleukin -6 (interleukin-6, IL-6) were overexpressed in the cell culture medium, and the effect of CUEDC2 on the TNF- alpha in acinar cells of acute pancreatitis was preliminarily discussed. Methods: 1 using rain frog hormone to induce acute pancreatitis The cell model.2 obtained the AR42J cells that overexpressed CUEDC2: the CUEDC2 plasmid was transferred into AR42J cells by liposome transfection, and the transfection efficiency.3 experimental grouping was examined by fluorescence microscopy: AR42J cells were divided into 4 groups: negative plasmid control group (Neg-Control group), negative plasmid rain frog group (Neg-CAE group), CUEDC2 plasmid control group (CUEDC2-Control) Group of CUEDC2 plasmids (group CUEDC2-CAE). The specific grouping method is as follows: negative plasmid group: transfection of negative plasmid to AR42J cell.Neg-CAE group to give 10-7 mol/L rain frog, Neg-Control group to give equal volume complete medium, and then divide two groups into 3 subgroups: 4h, 8h, 24h.CUEDC2 plasmid group: CUEDC2 plasmid transfection AR42J cell.CUEDC Group 2-CAE was given 10-7mol/L rain frog CUEDC2-Control group CUEDC2-Control group given equal volume complete medium, and then divided into 3 subgroups: 4h, 8 h, 24h.4 CUEDC2 determination: PCR detection CUEDC2 mRNA expression.Western-blot detection of CUEDC2 protein expression of amylase Se, AMY) level, the application of ELISA (enzyme linked immunosorbent assay) method to detect tumor necrosis factor alpha (tumor necrosis factor- a, TNF- alpha) and interleukin -6 (enzyme linked immunosorbent assay). An acute pancreatitis cell model was successfully established. The level of AMY in the rain frog group increased with time and was higher than that in the control group (P0.05). The CUEDC2mRNA level in the rain frog group was lower than that in the control group (P0.05). The CUEDC2mRNA of each time point in the rain frog group was not significantly changed (P0.05); 2 the green fluorescence was visible after the transfection of the CUEDC2 plasmid. The growth condition of the cells was good, which showed that the transfection condition was stable and the transfection was successful. The transfection rate was higher than that of the control group (P0.05), the transfection rate was about 65%-70%.CUEDC2 plasmid group (P0.05). The negative plasmid group had no significant difference compared with the control group (P0.05). The 3 cell culture supernatant AMY: was compared at every time point in the negative plasmid control group and CUEDC2 substance. There was no significant increase in the level of AMY in the control group, and there was no significant difference between the two groups (P0.05). There was no significant difference between the negative plasmid control group and the CUEDC2 plasmid control at the AMY level at each time point (P0.05). The AMY level increased gradually in the negative plasmid rain frog group and the CUEDC2 plasmid wet frog group (P0.05). The group and CUEDC2 plasmid wet frog group were all higher than those of the control group (P0.05), and the AMY level of the CUEDC2 plasmid wet frog group was lower than the negative plasmid (P0.05), and the 4 cell culture supernatant TNF- a, IL-6: in the negative plasmid control group and the CUEDC2 plasmid control group, the TNF- alpha, IL-6 level was lower, and there was no statistical significance between the two groups. (P0.05); the level of TNF- alpha and IL-6 increased gradually in the negative plasmids of the rain frog group and the CUEDC2 plasmid wet frog group (P0.05). The TNF- A and IL-6 levels of the negative plasmids and CUEDC2 plasmids were significantly higher than those of the control group at all time points (P0.05); CUEDC plasmid rain frog group and the negative plasmid, rain frog group. Compared with TNF- alpha at every time point, IL-6 water decreased (P0.05). Conclusion: 1 the AP cell model can be successfully prepared by the induction of the AP cell model.2 in the AP cell model, and the level of CUEDC2 mRNA in the AP cell model is downregulated and.3 successfully constructed the AP cell model for the expression of CUEDC2 protein, which lays a foundation for the study of the role of CEUDC2 in acute pancreatitis. After that, AR42J cells induced by rain frogs produced AMY, TNF- alpha and IL-6 levels decreased, indicating that CUEDC2 can inhibit the excessive growth of inflammatory mediators and its protective effect in acute pancreatitis.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R576

【参考文献】