CUEDC2对急性胰腺炎腺泡细胞凋亡的影响

发布时间：2018-03-14 02:33

本文选题：急性胰腺炎　切入点：CUEDC2　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：急性胰腺炎(Acute pancreatitis,AP)是由多种病因引起的急性炎症反应,具有起病急骤,发展迅速,病情较重,并发症较多等特点。临床上大多数患着为轻度急性胰腺炎,病程呈自限性,预后较好,重度急性胰腺炎常伴有胰腺及周边组织坏死,临床病情凶险,病死率高。AP的发病机制复杂,近年来人们发现细胞凋亡在AP的发生发展过程中起重要作用,凋亡和坏死是AP时胰腺腺泡细胞死亡的两种主要形式,二者最大本质区别是凋亡细胞形成凋亡小体后很快被巨噬细胞所吞噬,不引起或很少引起炎症反应,AP的严重性与坏死程度呈正相关,而与凋亡程度呈负相关。早期诱导腺泡细胞凋亡是治疗AP的关键环节。含CUE结构域蛋白2(CUE domain containing 2,CUEDC2)是近年来新发现的一种蛋白。CUEDC2可以抑制核因子-κB(nuclear factor-κB,NF-κB)信号通路和Janus激酶/信号转导和转录活化因子(Janus kinase/signal transducer and activator of tran-scriptions,JAK/STAT)信号通路,还能够抑制巨噬细胞的激活及热休克蛋白70的伴侣活性,CUEDC2也参与了细胞周期调节。文献研究发现CUEDC2在不同肿瘤中的表达水平及作用不同。同时,CUEDC2也参与了调控炎症反应,如梗阻性肾病、结肠炎相关肿瘤等。此外,CUEDC2还参与了心肌缺血再灌注损伤、慢性髓细胞白血病等细胞凋亡的相关研究。但目前尚无CUEDC2在急性胰腺炎中作用研究的报道,因而本研究从细胞凋亡的角度分析CUEDC2对急性胰腺炎腺泡细胞的影响。目的:应用雨蛙素干预AR42J细胞建立AP离体细胞模型,采用质粒转染技术对AR42J细胞CUEDC2进行过表达,探究CUEDC2对AP胰腺腺泡细胞凋亡的影响。方法:1应用雨蛙素干预AR42J细胞建立AP模型。应用质粒转染技术,构建过表达CUEDC2的AR42J细胞模型。2实验分组:(1)阴性质粒对照组(Neg-CON):转染p EGFP-N1质粒,完全培养基培养;(2)CUEDC2质粒对照组(CUEDC2-CON):转染p EGFP-N1-CUEDC2质粒,给予完全培养基培养;(3)阴性质粒雨蛙素组(Neg-CAE):转染p EGFP-N1质粒,给予含10-7 mmol/L雨蛙素培养基培养;(4)CUEDC2质粒雨蛙素组(CUEDC2-CAE):转染p EGFP-N1-CUEDC2质粒,给予含10-7 mmol/L雨蛙素培养基培养;再将细胞分为4h、8h、24h三个亚组。3应用RT-PCR检测CUEDC2 m RNA表达水平。应用Western blot检测CUEDC2、Bax和Bcl-2蛋白表达水平。Annexin V-PE/7-AAD双染联合流式细胞术检测造模后细胞早期凋亡率及晚期凋亡坏死率。结果:1 CUEDC2 m RNA表达:与对照组相比,雨蛙素组CUEDC2 m RNA表达量均降低,有统计学差异(P0.05),并且随着时间的进展,雨蛙素干预的各组间表达水平无差异(P0.05)。2建立过表达CUEDC2的AR42J细胞模型:比较各组CUEDC2蛋白表达水平,空白对照组和阴性质粒组之间无统计学差异(P0.05);CUEDC2质粒组表达高于其余两组,与其余两组比较均有统计学差异(P0.05)。可认为,成功建立了过表达CUEDC2的AR42J细胞模型。3 Bax的表达:与各自对照组相比,阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组Bax表达水平均增高(P0.05)。在两个对照组中,CUEDC2质粒对照组与阴性质粒对照组相比,Bax表达增高(P0.05),并且随着时间延长无明显变化。在两个雨蛙素组中,CUEDC2质粒雨蛙素组与阴性质粒雨蛙素组相比,4h、8h时Bax表达增高(P0.05),24h时无统计差异(P0.05),并且随着时间延长,阴性质粒雨蛙素组Bax表达逐渐增高,至8h最高,24h与8h无统计差异,而CUEDC2质粒雨蛙素组Bax表达与时间进展无相关性。4 Bcl-2的表达:与各自对照组相比,阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组Bcl-2表达水平均增高(P0.05)。在两个对照组中,CUEDC2质粒对照组与阴性质粒对照组相比,Bcl-2表达降低(P0.05),并且随着时间延长无明显变化。在两个雨蛙素组中,CUEDC2质粒雨蛙素组与阴性质粒雨蛙素组相比,4h、8h时Bcl-2表达降低(P0.05),24h时无统计差异(P0.05),并且随着时间延长,阴性质粒雨蛙素组Bcl-2表达逐渐降低,至8h最低,24h与8h无统计差异,而CUEDC2质粒雨蛙素组Bcl-2表达与时间进展无相关性。5 Bax/Bcl-2比值:与各自对照组相比,阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组Bax/Bcl-2比值均增高(P0.05)。在两个对照组中,CUEDC2质粒对照组与阴性质粒对照组相比,Bax/Bcl-2比值增高(P0.05),随着时间延长无明显变化。在两个雨蛙素组中,CUEDC2质粒雨蛙素组与阴性质粒雨蛙素组相比,4h、8h时Bax/Bcl-2比值增高(P0.05),24h时无统计差异(P0.05),并且随着时间延长,阴性质粒雨蛙素组Bax/Bcl-2比值逐渐增高,至8h最高,24h与8h无统计差异,而CUEDC2质粒雨蛙素组Bax/Bcl-2比值与时间进展无相关性。并且,阴性质粒对照组Bax/Bcl-2比值小于1,即Bcl-2相对较高,而CUEDC2过表达后或者雨蛙素干预后Bax/Bcl-2比值大于1,即Bax相对较高。6细胞早期凋亡率和晚期凋亡坏死率:与各自对照组相比,阴性质粒雨蛙素组和CUEDC2质粒雨蛙素组早期凋亡率和晚期凋亡坏死率均增高(P0.05)。在两个对照组中,CUEDC2质粒对照组与阴性质粒对照组相比,早期凋亡率增高,晚期凋亡坏死率降低(P0.05)。在两个雨蛙素组中,CUEDC2质粒雨蛙素组与阴性质粒雨蛙素组相比,8h时早期凋亡率增高,晚期凋亡坏死率降低(P0.05),而在24h时无统计差异(P0.05)。结论:1我们应用雨蛙素成功建立了AP模型,并通过质粒转染成功建立了过表达CUEDC2的AR42J细胞模型。2在AP细胞模型,CUEDC2 m RNA的表达水平下调。3在AP细胞模型,细胞凋亡和坏死率均增高,过表达CUEDC2后可在AP早期一定时间内促进细胞凋亡,减少部分细胞坏死,从而减轻AP。研究表明,CUEDC2可以促进胰腺腺泡细胞凋亡,在AP中是一种保护性蛋白。
[Abstract]:Acute pancreatitis (Acute pancreatitis AP) is an acute inflammation caused by a variety of causes, with abrupt onset, rapid development, severe illness, complications and other characteristics. Most cases with mild acute pancreatitis, disease is self limiting, good prognosis, severe acute pancreatitis associated with pancreatic necrosis and surrounding tissue. Clinical dangerous disease, pathogenesis of high mortality rate of.AP complex in recent years, people found that apoptosis play an important role in the development of AP, apoptosis and necrosis are two main forms of pancreatic acinar cell death AP, the two biggest difference is the essence of apoptotic cells and formation of apoptotic bodies soon engulfed by macrophages that does not cause or rarely cause inflammatory reaction, AP was positively correlated with the severity degree of necrosis, and negatively correlated with the degree of apoptosis. The early induction of apoptosis of acinar cells is the key to the treatment of AP Link. CUE domain containing protein 2 (CUE domain 2 containing, CUEDC2) is a recently discovered protein.CUEDC2 can inhibit nuclear factor kappa B (factor- K nuclear B NF- K B) signaling pathway and Janus kinase / signal transducer and activator of transcription (Janus kinase/signal transducer and activator of tran-scriptions. JAK/STAT) signaling pathway, but also can inhibit macrophage activation of heat shock protein 70 and chaperone activity, CUEDC2 is also involved in cell cycle regulation. The study found that the expression level and the role of CUEDC2 in different tumors are different. At the same time, CUEDC2 is also involved in the regulation of inflammation, such as obstructive nephropathy, colitis associated tumors in addition. CUEDC2, is also involved in myocardial ischemia reperfusion injury, apoptosis of chronic myeloid leukemia cells. But there is no research on the effect of CUEDC2 in acute pancreatitis is reported, because This study from the perspective of apoptosis of acinar cells in acute pancreatitis CUEDC2 analysis. The effects of caerulein intervention Objective: the application of AR42J cells to establish AP cell model in vitro, using plasmid transfection of AR42J cells by CUEDC2 overexpression, explore the effect of CUEDC2 on apoptosis of pancreatic acinar cells AP. Methods: 1 Application of caerulein based intervention the AP model of AR42J cells. Application of plasmid transfection technique, construct the over expression of AR42J cell model of.2 experimental group CUEDC2: (1) negative control plasmid group (Neg-CON): P transfected with EGFP-N1 plasmid, cultured; (2) CUEDC2 plasmid control group (CUEDC2-CON): P transfected with EGFP-N1-CUEDC2 plasmid, given the complete medium; (3) negative plasmid caerulein group (Neg-CAE): P transfected with EGFP-N1 plasmid, containing 10-7 mmol/L ceruletide medium; (4) CUEDC2 plasmid ceruletide group (CUEDC2-CAE): P transfected with EGFP-N1-CUEDC2 plasmid, to With caerulein containing 10-7 mmol/L medium; then the cells were divided into 4h, 8h, 24h expression levels of three subgroups of.3 detected by RT-PCR CUEDC2 m RNA. The application of Western blot detection CUEDC2, Bax expression and protein levels of Bcl-2.Annexin V-PE/7-AAD double staining combined with flow cytometry to detect early apoptosis and late apoptosis and necrosis rate after modeling the cells. Results: 1 CUEDC2 m RNA expression: compared with the control group, CUEDC2 M group of caerulein RNA expression was reduced, there were statistically significant differences (P0.05), and with the progress of time, each caerulein intervention expression had no difference (P0.05) to establish.2 cells overexpressing AR42J model CUEDC2 comparison of CUEDC2 protein expression level, between the control group and negative plasmid group showed no significant difference (P0.05); expression plasmid of CUEDC2 group was higher than that of the other two groups, and the remaining two groups were statistically significant (P0.05). It is considered that the success of construction The expression of AR42J.3 Bax cell model of CUEDC2: compared with the control group, negative plasmid group and CUEDC2 plasmid ceruletide caerulein group Bax expression levels were increased (P0.05). In two in the control group, CUEDC2 plasmid group compared with the negative control plasmid group, increased expression of Bax (P0.05) with the prolongation of time, and no significant changes. In two caerulein group, CUEDC2 plasmid group and negative plasmid ceruletide caerulein group compared to 4h, 8h increased the expression of Bax (P0.05), 24h had no statistical difference (P0.05), and with the prolongation of time, negative plasmid caerulein group the expression of Bax increased gradually to 8h, the highest, no statistical difference between 24h and 8h, the expression of CUEDC2 plasmid in caerulein group the expression of Bax and.4 have no correlation time of Bcl-2: compared with the control group, negative plasmid group and CUEDC2 plasmid ceruletide caerulein group Bcl-2 expression levels were increased in two (P0.05). In the control group, CUEDC2 plasmid group compared with the negative control plasmid group, decreased expression of Bcl-2 (P0.05), and with the prolongation of time without significant change. In two caerulein group, CUEDC2 plasmid group and negative plasmid ceruletide caerulein group, 4h, 8h Bcl-2 expression decreased (P0.05), 24h had no statistical difference (P0.05), and with the prolongation of time, negative plasmid caerulein group Bcl-2 expression gradually decreased to the lowest, 8h, no statistical difference between 24h and 8h, and the progress of CUEDC2 plasmid caerulein group the expression of Bcl-2 and.5 have no correlation time ratio of Bax/Bcl-2: compared with the control group, negative plasmid group and caerulein the CUEDC2 plasmid caerulein group Bax/Bcl-2 were increased (P0.05). In two in the control group, CUEDC2 plasmid group compared with the negative control plasmid group, (P0.05), Bax/Bcl-2 ratio increased with the prolongation of time without significant change. In two caerulein group, CUEDC2 Caerulein plasmid group and negative plasmid ceruletide group compared to 4h, increased Bax/Bcl-2 ratio 8h (P0.05), 24h had no statistical difference (P0.05), and with the prolongation of time, negative plasmid caerulein group Bax/Bcl-2 ratio gradually increased to the highest 8h, no statistical difference between 24h and 8h, and the progress of CUEDC2 plasmid ceruletide group Bax/Bcl-2 ratio and time correlation. And the negative control plasmid group Bax/Bcl-2 ratio is less than 1, Bcl-2 is relatively high, but after overexpression of CUEDC2 or caerulein intervention Bax/Bcl-2 ratio is greater than 1, which is relatively high Bax.6 cell early apoptosis rate and the late apoptosis and necrosis rate: compared with the control group, negative plasmid hylid peptide group and CUEDC2 plasmid group ceruletide apoptotic rate and necrosis rate increased (P0.05). In two in the control group, CUEDC2 plasmid group compared with the negative control plasmid group, early apoptosis rate increased, apoptosis The necrosis rate decreased (P0.05). In two caerulein group, CUEDC2 plasmid group and negative plasmid ceruletide caerulein compared 8h early apoptosis rate increased, the late apoptosis and necrosis rate decreased (P0.05), but no statistical difference in 24h (P0.05). Conclusion: 1 we successfully established the application of caerulein the AP model, and the plasmid was successfully established over expression of.2 AR42J in AP cell model CUEDC2 cell model, the expression level of CUEDC2 m RNA.3 was down regulated in AP cell model, cell apoptosis and necrosis rate were increased after overexpression of CUEDC2 can induce cell apoptosis in the early stage of AP within a certain period of time, reduce the cell necrosis in order to reduce, AP. research shows that CUEDC2 can promote the apoptosis of pancreatic acinar cells in AP is a kind of protective protein.

【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

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