本文選題：胰島再生源蛋白I + 胰島再生源蛋白III�。� 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：重癥急性胰腺炎（severe acute pancreatitis，SAP）是臨床常見的急腹癥，其發(fā)病急、病情進(jìn)展快，并發(fā)癥多，目前病死率仍高達(dá)30％。近幾年研究顯示SAP時(shí)腸黏膜屏障功能受損，容易發(fā)生腸屏障功能障礙(intestine barrier functional disturbance，IBFD)，使腸黏膜通透性增加，是導(dǎo)致腸道細(xì)菌及內(nèi)毒素發(fā)生移位，加速敗血癥的進(jìn)程，，誘發(fā)和加重全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome,SIRS)、多器官功能障礙綜合癥(Multiple organ dysfunction syndrome,MODS)，甚至?xí)鹚劳觯虼嗽谥委熢l(fā)病的同時(shí)對(duì)腸黏膜屏障的保護(hù)至關(guān)重要。近年來胰島再生源蛋白(regenerating islet-derived protein, Reg)在SAP發(fā)生發(fā)展過程中的作用日益引起廣泛關(guān)注。 Reg蛋白是近幾年發(fā)現(xiàn)的一類多功能分子，屬C型凝集素超家族，大量研究表明Reg蛋白在多種生理、病理活動(dòng)中發(fā)揮重要作用，在SAP發(fā)生發(fā)展過程中與促進(jìn)細(xì)胞增殖、調(diào)控小學(xué)校細(xì)胞凋亡、抑制炎癥因子過表達(dá)及抑制損傷部位周圍病原微生物生長(zhǎng)和擴(kuò)散等功能有關(guān)，尤其再生基因RegI、III在急性胰腺炎（acute pancreatitis，AP）中的作用日益受到重視，現(xiàn)已有研究表明Reg可通過抑制NF-κB入核來調(diào)節(jié)TNF-α、IL-6等誘導(dǎo)的炎癥因子的過表達(dá)從而抑制炎癥的發(fā)生，還有研究顯示IL-22可以調(diào)節(jié)Reg的表達(dá)，來抑制損傷部位細(xì)菌生長(zhǎng)，但其在SAP時(shí)腸黏膜損害中的作用研究甚少。 本實(shí)驗(yàn)旨在通過測(cè)定RegI、III蛋白及mRNA在SAP大鼠小腸中的表達(dá)，評(píng)價(jià)RegI、III水平與腸黏膜屏障損傷的關(guān)系；并通過分析應(yīng)用特異性NF-κB抑制劑吡咯烷二硫代氨基甲酸鹽（pyrrolidine dithiocarbamate，PDTC）預(yù)處理后的小腸RegI、III的變化，初步探討Reg蛋白家族與NF-κB通路的關(guān)系。 方法：1實(shí)驗(yàn)分組120只成年SD大鼠分為對(duì)照組（N組）、重癥急性胰腺組（S組）、1mg/kg PDTC+重癥急性胰腺炎組（P1組）、10mg/kgPDTC+重癥急性胰腺炎組（P10組）、100mg/kgPDTC+重癥急性胰腺炎組（P100組），各為12小時(shí)及24小時(shí)兩組，共10組每組12只大鼠。 2制造模型：本研究采20%L-精氨酸腹腔注射，間隔一小時(shí)制作大鼠SAP模型。S組腹腔注射20%L-精氨酸2.5g/kg2次（總共5g/kg大鼠質(zhì)量），間隔一小時(shí)，誘導(dǎo)重癥急性胰腺炎模型；N組于腹腔注射等體積0.9%氯化鈉；P1、P10、P100組：于造模前1小時(shí)腹腔注射PDTC1mg/kg、10mg/kg、100mg/kg預(yù)處理。各組造模成功后12小時(shí)及24小時(shí)取血、胰腺及小腸組織。 3實(shí)驗(yàn)方法：開腹后觀察胰腺、腸道及周圍組織情況，并通過蘇木素-伊紅（htoxylin eosin HE）染色光學(xué)顯微鏡觀察胰腺、小腸的病理變化，ELISA方法檢測(cè)血清中IL-22、TNF-α及I-FABP水平，采用熒光定量RT-PCR測(cè)定小腸組織中RegI、IIImRNA表達(dá)含量，應(yīng)用Western blot檢測(cè)小腸組織中NF-κB p65及RegI、III蛋白水平。 4統(tǒng)計(jì)學(xué)方法：應(yīng)用SPSS17.0軟件包進(jìn)行統(tǒng)計(jì)學(xué)處理。設(shè)α=0.05，P0.05為差異顯著，具有統(tǒng)計(jì)學(xué)意義。 結(jié)果：1HE染色，S組胰腺組織結(jié)構(gòu)紊亂，間質(zhì)水腫明顯，可見腺泡細(xì)胞壞死及出血，炎性細(xì)胞浸潤(rùn)，并隨著時(shí)間進(jìn)展而漸進(jìn)性加重。腸道HE染色S組上皮細(xì)胞層壞死、脫落，部分絨毛形態(tài)不規(guī)則、部分脫落，固有層崩解，毛細(xì)血管擴(kuò)張、充血，炎癥細(xì)胞的浸潤(rùn)，24h較12h更嚴(yán)重。胰腺病理采用Schmidt評(píng)分、腸黏膜病理采用Chiu評(píng)分。結(jié)果顯示S組較N組明顯升高（P0.01），且24h比12h更加嚴(yán)重（P0.05），提示L-精氨酸可成功誘導(dǎo)重癥急性胰腺炎大鼠腸黏膜屏障損傷模型。 2應(yīng)用不同劑量PDTC干預(yù)后發(fā)現(xiàn)無論12h或24h，P1及P10組大鼠胰腺及腸道組織病理評(píng)分與S組比較明顯降低（P0.05）, P100組與S組比較胰腺及腸道組織評(píng)分無明顯差異（P0.05）；P10組在12h腸道評(píng)分較P1組無明顯差異（P0.05）,而24hP10組評(píng)分較P1可見減低（P0.05）；P100組較P1組無論12h或24h均有明顯升高（P0.05）；腸道評(píng)分S、P124h組較12h組明顯升高（P0.05）。 3血清IL-22：S組明顯高于N組（P0.01）；給予PDTC干預(yù)后，PDTC不同劑量組與S組比較明顯降低（P0.05）；與P1組比較，12h及24h P10組無明顯變化（P0.05），而P100組較P1組可見明顯升高（P0.05）；與P10組比較，P100組表達(dá)上升（P0.05）；S組24h IL-22較12h明顯升高（P0.05）。 4血清TNF-α：與N組比較S組TNF-α表達(dá)明顯升高（P0.01）；應(yīng)用PDTC干預(yù)后，與S組比較P1、P10組表達(dá)降低（P0.05），而P100組較S組無明顯變化（P0.05）；P10組TNF-α表達(dá)低于P1組（P0.05），而P100組表達(dá)高于P1組（P0.05）；S組、P100組24h較12h有明顯升高（P0.05）。 5血清I-FABP：S組I-FABP表達(dá)明顯高于N組（P0.01）；應(yīng)用不同劑量PDTC干預(yù)后，與S組比較P1、P10組表達(dá)降低（P0.01），而P100組較S組無明顯變化（P0.05）；P10組I-FABP表達(dá)低于P1組（P0.05），而P100組表達(dá)高于P1組（P0.05）；S組24h較12h有明顯升高（P0.05）。 6小腸NF-κBp65：與N組比較S組NF-κB p65表達(dá)明顯升高（P0.01）；給予PDTC干預(yù)后，P1及P10組與SAP組比較表達(dá)降低（P0.05），而P100組較S組無明顯變化（P0.05）；P10組NF-κB p65表達(dá)低于P1組（P0.05），而P100組表達(dá)高于P1組（P0.05）；S組24h較12h NF-κB p65表達(dá)有明顯升高（P0.05）。 7小腸RegI、III蛋白：S組RegI、III蛋白表達(dá)明顯均高于N組（P0.01）；應(yīng)用不同劑量PDTC干預(yù)后，與S組比較，P1、P10組表達(dá)降低（P0.01），而P100組較S組無明顯變化（P0.05）；P10組RegI、III蛋白低于P1組（P0.05），而P100組表達(dá)高于P1組（P0.01）； RegI、III蛋白S及P100組24h較12h有明顯升高（P0.05）。 8小腸RegI、III mRNA：與N組比較S組RegI、III mRNA表達(dá)明顯升高（P0.05）；應(yīng)用PDTC干預(yù)后，P1及P10組與S組比較表達(dá)降低（P0.05），而P100組較S組無明顯變化（P0.05）；P10組RegIII mRNA低于P1組（P0.05），而P100組表達(dá)高于P1組（P0.05）；而小腸組織中P10組RegI mRNA表達(dá)12h較P1組明顯降低（P0.05），24h較P1組無統(tǒng)計(jì)學(xué)差異（P0.05），在24及12h均可見P100組高于P10組（P0.05）；S24h組較12h明顯升高（P0.05）。 9RegI、III蛋白表達(dá)與腸黏膜病理評(píng)評(píng)分、IL-22、I-FABP、TNF-α及NF-κBp65表達(dá)呈正相關(guān)。 結(jié)論：1SAP大鼠小腸RegI、III表達(dá)增高，考慮RegI、III為腸黏膜屏障中調(diào)節(jié)因子之一，且其表達(dá)可能與IL-22、TNF-α等炎癥因子有關(guān)。 2PDTC對(duì)于NF-κB通路抑制作用與劑量相關(guān)，1mg/kg、10mg/kgPDTC抑制NF-κB通路活性，減輕SAP大鼠胰腺及腸屏障損傷，以10mg/kg作用更為明顯，100mg/kg PDTC不能減輕胰腺及腸屏障損傷，但不論12h或24h100mg/kg PDTC組各指標(biāo)均未達(dá)到SAP組水平。 3RegI、III基因表達(dá)可部分通過NF-κB通路調(diào)節(jié)，腸黏膜損傷或炎癥因子刺激致RegI、III表達(dá)上調(diào)，對(duì)黏膜損傷起起作用，其作用機(jī)制考慮為多個(gè)通路的共同調(diào)節(jié)，且不除外RegI、III基因之間的存在協(xié)同或拮抗作用，對(duì)于過度表達(dá)的RegI、III是否會(huì)加劇SAP時(shí)的腸黏膜損傷仍需進(jìn)一步實(shí)驗(yàn)證實(shí)。
[Abstract]:Objective: severe acute pancreatitis (SAP) is a common clinical acute abdomen. It has a rapid onset, rapid progression and many complications. The current mortality rate is still up to 30%. in recent years, which shows that the intestinal mucosal barrier function is damaged and the intestinal barrier function is easily impaired (intestine barrier functional disturbance, IBFD) in SAP. The increase of intestinal mucosal permeability is the cause of intestinal bacteria and endotoxin translocation, accelerating the process of septicemia, inducing and aggravating the systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), multiple organ dysfunction syndrome (Multiple organ dysfunction syndrome, MODS), and even cause death, therefore in treatment In recent years, the role of regenerating islet-derived protein (Reg) in the development of SAP has attracted more and more attention.
Reg protein is a kind of multifunctional molecule found in recent years, belonging to C type lectin superfamily. A large number of studies have shown that Reg protein plays an important role in a variety of physiological and pathological activities. In the process of SAP development, it can promote cell proliferation, regulate primary school cell apoptosis, inhibit the overexpression of inflammatory factors and inhibit the pathogenic microbiology around the site of injury. The function of growth and diffusion, especially the function of regenerated gene RegI, III in acute pancreatitis (AP), has been paid more and more attention. Now there have been studies showing that Reg can inhibit the overexpression of the inflammatory factors induced by TNF- a, IL-6 and so on by inhibiting NF- kappa B into the nucleus to inhibit the occurrence of inflammation, and the study shows that IL-22 can be adjusted. The expression of Reg inhibits the growth of bacteria in the injured area, but little is known about its role in intestinal mucosal damage at SAP.
The purpose of this study was to evaluate the relationship between the expression of RegI, III protein and mRNA in the small intestine of SAP rats, and to evaluate the relationship between the level of RegI, III and the damage of intestinal mucosal barrier, and to discuss the changes of small intestine RegI after the application of the specific NF- kappa B inhibitor pyrrolidine two thiocarbamate (pyrrolidine dithiocarbamate, PDTC). The relationship between the eg protein family and the NF- kappa B pathway.
Methods: 1 experimental group 120 adult SD rats were divided into control group (group N), severe acute pancreas group (group S), 1mg/kg PDTC+ severe acute pancreatitis group (group P1), 10mg/kgPDTC+ severe acute pancreatitis group (P10 group), 100mg/kgPDTC+ severe acute pancreatitis group (P100 group), each 12 hours and 24 hours two groups, a total of 10 groups of 12 rats.
2 manufacturing model: 20%L- arginine was injected intraperitoneally in this study, and one hour interval was made to make rat SAP model.S group 20%L- arginine 2.5g/kg2 times 2.5g/kg2 (total 5g/kg rat mass) and interval of one hour to induce severe acute pancreatitis model; group N was injected with equal volume of sodium chloride in abdominal cavity; P1, P10, P100 group: abdominal cavity 1 hours before the model. PDTC1mg/kg, 10mg/kg and 100mg/kg were injected into each group. Blood, pancreas and small intestine were collected 12 hours and 24 hours after successful modeling.
3 experimental methods: To observe the condition of pancreas, intestines and surrounding tissues after laparotomy, and observe the pathological changes of pancreas and small intestine by htoxylin eosin HE staining optical microscope. ELISA method was used to detect the level of IL-22, TNF- alpha and I-FABP in serum. The expression of RegI and IIImRNA in small intestine was measured by fluorescence quantitative RT-PCR, and Wes was used in Wes. Tern blot was used to detect the levels of NF- - B p65 and RegI and III protein in the small intestine.
4 statistical method: SPSS17.0 software package was used for statistical analysis. Setting alpha =0.05 and P0.05 was statistically significant.
Results: 1HE staining, in group S, the structure of the pancreas was disturbed and the interstitial edema was obvious. The acinar cells were necrotic and bleeding, and inflammatory cells infiltrated, and gradually increased with the progress of time. The epithelial cell layer of the intestinal HE staining S group was necrotic, shedding, irregular, partial disintegration, lamina propria, capillary dilatation, congestion, and inflammation. The invasion of cell was more serious than that of 12h. The pathology of pancreas was Schmidt score, and the pathology of intestinal mucosa was Chiu score. The results showed that the S group was significantly higher than the N group (P0.01), and 24h was more serious than 12h (P0.05), suggesting that L- arginine could successfully induce the intestinal mucosal barrier damage model of severe acute pancreatitis in rats.
2 after the intervention of different doses of PDTC, the pathological scores of pancreas and intestinal tissue in group P1 and P10 group were significantly lower than those in group S (P0.05). There was no significant difference in pancreas and intestinal tissue scores in P100 and S groups (P0.05), and there was no significant difference in 12h intestinal score between P100 and S groups (P0.05). Low (P0.05); P100 group was significantly higher than group P1 (12h or 24h) (P0.05); intestinal score S, P124h group was significantly higher than 12h group (P0.05).
3 the serum IL-22:S group was significantly higher than the N group (P0.01), and the prognosis of PDTC was significantly lower than that of the S group (P0.05). Compared with the P1 group, there was no obvious change in 12h and 24h P10 group (P0.05). 05).
4 serum TNF- alpha: the expression of TNF- alpha in group S was significantly higher than that in group N (P0.01). The expression of PDTC in group S was lower than that in S group (P0.05), while P100 group was lower than that of S group.
5 the expression of I-FABP in the serum I-FABP:S group was significantly higher than that in the N group (P0.01), and the expression of P10 group was lower (P0.01) compared with the S group (P0.01), and the P100 group was less than the S group (P0.05) compared with the S group, and the expression was higher than that in the S group (P0.05).
6 small intestine NF- kappa Bp65: compared with group N, the expression of NF- kappa B p65 increased significantly in group S (P0.01), and the expression of P1 and P10 group was lower than that of SAP group, while the group of P1 and P10 was less than that of the SAP group. Increase (P0.05).
7 RegI, III protein: S group RegI, III protein expression was significantly higher than that of N group (P0.01), and the expression of P1, P10 group was lower than that of S group. It was significantly higher than 12h (P0.05).
8 small intestine RegI, III mRNA: the expression of III mRNA in S group was significantly higher than that in group N (P0.05), and the expression of P1 and P10 groups was lower than that in the S group. 12h was significantly lower than that in group P1 (P0.05), and 24h was not significantly different from P1 group (P0.05). In 24 and 12h, P100 group was higher than P10 group (P0.05); S24h group was significantly higher than that of P10 group.
The expression of 9RegI and III protein was positively correlated with Intestinal Mucosal pathological grading, IL-22, I-FABP, TNF- alpha and NF- kappa Bp65 expression.
Conclusion: the expression of RegI and III in the small intestine of 1SAP rats is higher. Considering RegI, III is one of the regulatory factors in the intestinal mucosal barrier, and its expression may be related to the inflammatory factors such as IL-22, TNF- a.
The inhibitory effect of 2PDTC on the NF- kappa B pathway is related to the dose. 1mg/kg, 10mg/kgPDTC inhibits the activity of NF- kappa B pathway, reduces the injury of the pancreatic and intestinal barrier in SAP rats, and is more obvious in 10mg/kg. 100mg/kg PDTC can not reduce the injury of the pancreas and intestinal barrier.
3RegI, III gene expression can be regulated partly through the NF- kappa B pathway. Intestinal mucosal injury or inflammatory factor stimulates RegI, III expression is up-regulated, and it plays a role in mucosal damage. The mechanism of action is considered as a common regulation of multiple pathways, without the exception of RegI, III genes are co or antagonistic to the overexpressed RegI, III will be added. The injury of intestinal mucosa in SAP is still need further experimental confirmation.
【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】：2014
【分類號(hào)】：R576

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