本文選題：急性胰腺炎　切入點：CUEDC2　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：急性胰腺炎(Acute pancreatitis,AP)是由多種病因引起的急性炎癥反應(yīng),具有起病急驟,發(fā)展迅速,病情較重,并發(fā)癥較多等特點。臨床上大多數(shù)患著為輕度急性胰腺炎,病程呈自限性,預(yù)后較好,重度急性胰腺炎常伴有胰腺及周邊組織壞死,臨床病情兇險,病死率高。AP的發(fā)病機制復(fù)雜,近年來人們發(fā)現(xiàn)細(xì)胞凋亡在AP的發(fā)生發(fā)展過程中起重要作用,凋亡和壞死是AP時胰腺腺泡細(xì)胞死亡的兩種主要形式,二者最大本質(zhì)區(qū)別是凋亡細(xì)胞形成凋亡小體后很快被巨噬細(xì)胞所吞噬,不引起或很少引起炎癥反應(yīng),AP的嚴(yán)重性與壞死程度呈正相關(guān),而與凋亡程度呈負(fù)相關(guān)。早期誘導(dǎo)腺泡細(xì)胞凋亡是治療AP的關(guān)鍵環(huán)節(jié)。含CUE結(jié)構(gòu)域蛋白2(CUE domain containing 2,CUEDC2)是近年來新發(fā)現(xiàn)的一種蛋白。CUEDC2可以抑制核因子-κB(nuclear factor-κB,NF-κB)信號通路和Janus激酶/信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子(Janus kinase/signal transducer and activator of tran-scriptions,JAK/STAT)信號通路,還能夠抑制巨噬細(xì)胞的激活及熱休克蛋白70的伴侶活性,CUEDC2也參與了細(xì)胞周期調(diào)節(jié)。文獻(xiàn)研究發(fā)現(xiàn)CUEDC2在不同腫瘤中的表達(dá)水平及作用不同。同時,CUEDC2也參與了調(diào)控炎癥反應(yīng),如梗阻性腎病、結(jié)腸炎相關(guān)腫瘤等。此外,CUEDC2還參與了心肌缺血再灌注損傷、慢性髓細(xì)胞白血病等細(xì)胞凋亡的相關(guān)研究。但目前尚無CUEDC2在急性胰腺炎中作用研究的報道,因而本研究從細(xì)胞凋亡的角度分析CUEDC2對急性胰腺炎腺泡細(xì)胞的影響。目的:應(yīng)用雨蛙素干預(yù)AR42J細(xì)胞建立AP離體細(xì)胞模型,采用質(zhì)粒轉(zhuǎn)染技術(shù)對AR42J細(xì)胞CUEDC2進(jìn)行過表達(dá),探究CUEDC2對AP胰腺腺泡細(xì)胞凋亡的影響。方法:1應(yīng)用雨蛙素干預(yù)AR42J細(xì)胞建立AP模型。應(yīng)用質(zhì)粒轉(zhuǎn)染技術(shù),構(gòu)建過表達(dá)CUEDC2的AR42J細(xì)胞模型。2實驗分組:(1)陰性質(zhì)粒對照組(Neg-CON):轉(zhuǎn)染p EGFP-N1質(zhì)粒,完全培養(yǎng)基培養(yǎng);(2)CUEDC2質(zhì)粒對照組(CUEDC2-CON):轉(zhuǎn)染p EGFP-N1-CUEDC2質(zhì)粒,給予完全培養(yǎng)基培養(yǎng);(3)陰性質(zhì)粒雨蛙素組(Neg-CAE):轉(zhuǎn)染p EGFP-N1質(zhì)粒,給予含10-7 mmol/L雨蛙素培養(yǎng)基培養(yǎng);(4)CUEDC2質(zhì)粒雨蛙素組(CUEDC2-CAE):轉(zhuǎn)染p EGFP-N1-CUEDC2質(zhì)粒,給予含10-7 mmol/L雨蛙素培養(yǎng)基培養(yǎng);再將細(xì)胞分為4h、8h、24h三個亞組。3應(yīng)用RT-PCR檢測CUEDC2 m RNA表達(dá)水平。應(yīng)用Western blot檢測CUEDC2、Bax和Bcl-2蛋白表達(dá)水平。Annexin V-PE/7-AAD雙染聯(lián)合流式細(xì)胞術(shù)檢測造模后細(xì)胞早期凋亡率及晚期凋亡壞死率。結(jié)果:1 CUEDC2 m RNA表達(dá):與對照組相比,雨蛙素組CUEDC2 m RNA表達(dá)量均降低,有統(tǒng)計學(xué)差異(P0.05),并且隨著時間的進(jìn)展,雨蛙素干預(yù)的各組間表達(dá)水平無差異(P0.05)。2建立過表達(dá)CUEDC2的AR42J細(xì)胞模型:比較各組CUEDC2蛋白表達(dá)水平,空白對照組和陰性質(zhì)粒組之間無統(tǒng)計學(xué)差異(P0.05);CUEDC2質(zhì)粒組表達(dá)高于其余兩組,與其余兩組比較均有統(tǒng)計學(xué)差異(P0.05)�？烧J(rèn)為,成功建立了過表達(dá)CUEDC2的AR42J細(xì)胞模型。3 Bax的表達(dá):與各自對照組相比,陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組Bax表達(dá)水平均增高(P0.05)。在兩個對照組中,CUEDC2質(zhì)粒對照組與陰性質(zhì)粒對照組相比,Bax表達(dá)增高(P0.05),并且隨著時間延長無明顯變化。在兩個雨蛙素組中,CUEDC2質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比,4h、8h時Bax表達(dá)增高(P0.05),24h時無統(tǒng)計差異(P0.05),并且隨著時間延長,陰性質(zhì)粒雨蛙素組Bax表達(dá)逐漸增高,至8h最高,24h與8h無統(tǒng)計差異,而CUEDC2質(zhì)粒雨蛙素組Bax表達(dá)與時間進(jìn)展無相關(guān)性。4 Bcl-2的表達(dá):與各自對照組相比,陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組Bcl-2表達(dá)水平均增高(P0.05)。在兩個對照組中,CUEDC2質(zhì)粒對照組與陰性質(zhì)粒對照組相比,Bcl-2表達(dá)降低(P0.05),并且隨著時間延長無明顯變化。在兩個雨蛙素組中,CUEDC2質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比,4h、8h時Bcl-2表達(dá)降低(P0.05),24h時無統(tǒng)計差異(P0.05),并且隨著時間延長,陰性質(zhì)粒雨蛙素組Bcl-2表達(dá)逐漸降低,至8h最低,24h與8h無統(tǒng)計差異,而CUEDC2質(zhì)粒雨蛙素組Bcl-2表達(dá)與時間進(jìn)展無相關(guān)性。5 Bax/Bcl-2比值:與各自對照組相比,陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組Bax/Bcl-2比值均增高(P0.05)。在兩個對照組中,CUEDC2質(zhì)粒對照組與陰性質(zhì)粒對照組相比,Bax/Bcl-2比值增高(P0.05),隨著時間延長無明顯變化。在兩個雨蛙素組中,CUEDC2質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比,4h、8h時Bax/Bcl-2比值增高(P0.05),24h時無統(tǒng)計差異(P0.05),并且隨著時間延長,陰性質(zhì)粒雨蛙素組Bax/Bcl-2比值逐漸增高,至8h最高,24h與8h無統(tǒng)計差異,而CUEDC2質(zhì)粒雨蛙素組Bax/Bcl-2比值與時間進(jìn)展無相關(guān)性。并且,陰性質(zhì)粒對照組Bax/Bcl-2比值小于1,即Bcl-2相對較高,而CUEDC2過表達(dá)后或者雨蛙素干預(yù)后Bax/Bcl-2比值大于1,即Bax相對較高。6細(xì)胞早期凋亡率和晚期凋亡壞死率:與各自對照組相比,陰性質(zhì)粒雨蛙素組和CUEDC2質(zhì)粒雨蛙素組早期凋亡率和晚期凋亡壞死率均增高(P0.05)。在兩個對照組中,CUEDC2質(zhì)粒對照組與陰性質(zhì)粒對照組相比,早期凋亡率增高,晚期凋亡壞死率降低(P0.05)。在兩個雨蛙素組中,CUEDC2質(zhì)粒雨蛙素組與陰性質(zhì)粒雨蛙素組相比,8h時早期凋亡率增高,晚期凋亡壞死率降低(P0.05),而在24h時無統(tǒng)計差異(P0.05)。結(jié)論:1我們應(yīng)用雨蛙素成功建立了AP模型,并通過質(zhì)粒轉(zhuǎn)染成功建立了過表達(dá)CUEDC2的AR42J細(xì)胞模型。2在AP細(xì)胞模型,CUEDC2 m RNA的表達(dá)水平下調(diào)。3在AP細(xì)胞模型,細(xì)胞凋亡和壞死率均增高,過表達(dá)CUEDC2后可在AP早期一定時間內(nèi)促進(jìn)細(xì)胞凋亡,減少部分細(xì)胞壞死,從而減輕AP。研究表明,CUEDC2可以促進(jìn)胰腺腺泡細(xì)胞凋亡,在AP中是一種保護(hù)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R576

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