Reg蛋白在重症急性胰腺炎大鼠肠黏膜屏障中的作用及其机制的研究

发布时间：2018-06-30 16:49

本文选题：胰岛再生源蛋白I + 胰岛再生源蛋白III　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：重症急性胰腺炎（severe acute pancreatitis，SAP）是临床常见的急腹症，其发病急、病情进展快，并发症多，目前病死率仍高达30％。近几年研究显示SAP时肠黏膜屏障功能受损，容易发生肠屏障功能障碍(intestine barrier functional disturbance，IBFD)，使肠黏膜通透性增加，是导致肠道细菌及内毒素发生移位，加速败血症的进程，，诱发和加重全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)、多器官功能障碍综合症(Multiple organ dysfunction syndrome,MODS)，甚至会引起死亡，因此在治疗原发病的同时对肠黏膜屏障的保护至关重要。近年来胰岛再生源蛋白(regenerating islet-derived protein, Reg)在SAP发生发展过程中的作用日益引起广泛关注。 Reg蛋白是近几年发现的一类多功能分子，属C型凝集素超家族，大量研究表明Reg蛋白在多种生理、病理活动中发挥重要作用，在SAP发生发展过程中与促进细胞增殖、调控小学校细胞凋亡、抑制炎症因子过表达及抑制损伤部位周围病原微生物生长和扩散等功能有关，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视，现已有研究表明Reg可通过抑制NF-κB入核来调节TNF-α、IL-6等诱导的炎症因子的过表达从而抑制炎症的发生，还有研究显示IL-22可以调节Reg的表达，来抑制损伤部位细菌生长，但其在SAP时肠黏膜损害中的作用研究甚少。本实验旨在通过测定RegI、III蛋白及mRNA在SAP大鼠小肠中的表达，评价RegI、III水平与肠黏膜屏障损伤的关系；并通过分析应用特异性NF-κB抑制剂吡咯烷二硫代氨基甲酸盐（pyrrolidine dithiocarbamate，PDTC）预处理后的小肠RegI、III的变化，初步探讨Reg蛋白家族与NF-κB通路的关系。方法：1实验分组120只成年SD大鼠分为对照组（N组）、重症急性胰腺组（S组）、1mg/kg PDTC+重症急性胰腺炎组（P1组）、10mg/kgPDTC+重症急性胰腺炎组（P10组）、100mg/kgPDTC+重症急性胰腺炎组（P100组），各为12小时及24小时两组，共10组每组12只大鼠。 2制造模型：本研究采20%L-精氨酸腹腔注射，间隔一小时制作大鼠SAP模型。S组腹腔注射20%L-精氨酸2.5g/kg2次（总共5g/kg大鼠质量），间隔一小时，诱导重症急性胰腺炎模型；N组于腹腔注射等体积0.9%氯化钠；P1、P10、P100组：于造模前1小时腹腔注射PDTC1mg/kg、10mg/kg、100mg/kg预处理。各组造模成功后12小时及24小时取血、胰腺及小肠组织。 3实验方法：开腹后观察胰腺、肠道及周围组织情况，并通过苏木素-伊红（htoxylin eosin HE）染色光学显微镜观察胰腺、小肠的病理变化，ELISA方法检测血清中IL-22、TNF-α及I-FABP水平，采用荧光定量RT-PCR测定小肠组织中RegI、IIImRNA表达含量，应用Western blot检测小肠组织中NF-κB p65及RegI、III蛋白水平。 4统计学方法：应用SPSS17.0软件包进行统计学处理。设α=0.05，P0.05为差异显著，具有统计学意义。结果：1HE染色，S组胰腺组织结构紊乱，间质水肿明显，可见腺泡细胞坏死及出血，炎性细胞浸润，并随着时间进展而渐进性加重。肠道HE染色S组上皮细胞层坏死、脱落，部分绒毛形态不规则、部分脱落，固有层崩解，毛细血管扩张、充血，炎症细胞的浸润，24h较12h更严重。胰腺病理采用Schmidt评分、肠黏膜病理采用Chiu评分。结果显示S组较N组明显升高（P0.01），且24h比12h更加严重（P0.05），提示L-精氨酸可成功诱导重症急性胰腺炎大鼠肠黏膜屏障损伤模型。 2应用不同剂量PDTC干预后发现无论12h或24h，P1及P10组大鼠胰腺及肠道组织病理评分与S组比较明显降低（P0.05）, P100组与S组比较胰腺及肠道组织评分无明显差异（P0.05）；P10组在12h肠道评分较P1组无明显差异（P0.05）,而24hP10组评分较P1可见减低（P0.05）；P100组较P1组无论12h或24h均有明显升高（P0.05）；肠道评分S、P124h组较12h组明显升高（P0.05）。 3血清IL-22：S组明显高于N组（P0.01）；给予PDTC干预后，PDTC不同剂量组与S组比较明显降低（P0.05）；与P1组比较，12h及24h P10组无明显变化（P0.05），而P100组较P1组可见明显升高（P0.05）；与P10组比较，P100组表达上升（P0.05）；S组24h IL-22较12h明显升高（P0.05）。 4血清TNF-α：与N组比较S组TNF-α表达明显升高（P0.01）；应用PDTC干预后，与S组比较P1、P10组表达降低（P0.05），而P100组较S组无明显变化（P0.05）；P10组TNF-α表达低于P1组（P0.05），而P100组表达高于P1组（P0.05）；S组、P100组24h较12h有明显升高（P0.05）。 5血清I-FABP：S组I-FABP表达明显高于N组（P0.01）；应用不同剂量PDTC干预后，与S组比较P1、P10组表达降低（P0.01），而P100组较S组无明显变化（P0.05）；P10组I-FABP表达低于P1组（P0.05），而P100组表达高于P1组（P0.05）；S组24h较12h有明显升高（P0.05）。 6小肠NF-κBp65：与N组比较S组NF-κB p65表达明显升高（P0.01）；给予PDTC干预后，P1及P10组与SAP组比较表达降低（P0.05），而P100组较S组无明显变化（P0.05）；P10组NF-κB p65表达低于P1组（P0.05），而P100组表达高于P1组（P0.05）；S组24h较12h NF-κB p65表达有明显升高（P0.05）。 7小肠RegI、III蛋白：S组RegI、III蛋白表达明显均高于N组（P0.01）；应用不同剂量PDTC干预后，与S组比较，P1、P10组表达降低（P0.01），而P100组较S组无明显变化（P0.05）；P10组RegI、III蛋白低于P1组（P0.05），而P100组表达高于P1组（P0.01）； RegI、III蛋白S及P100组24h较12h有明显升高（P0.05）。 8小肠RegI、III mRNA：与N组比较S组RegI、III mRNA表达明显升高（P0.05）；应用PDTC干预后，P1及P10组与S组比较表达降低（P0.05），而P100组较S组无明显变化（P0.05）；P10组RegIII mRNA低于P1组（P0.05），而P100组表达高于P1组（P0.05）；而小肠组织中P10组RegI mRNA表达12h较P1组明显降低（P0.05），24h较P1组无统计学差异（P0.05），在24及12h均可见P100组高于P10组（P0.05）；S24h组较12h明显升高（P0.05）。 9RegI、III蛋白表达与肠黏膜病理评评分、IL-22、I-FABP、TNF-α及NF-κBp65表达呈正相关。结论：1SAP大鼠小肠RegI、III表达增高，考虑RegI、III为肠黏膜屏障中调节因子之一，且其表达可能与IL-22、TNF-α等炎症因子有关。 2PDTC对于NF-κB通路抑制作用与剂量相关，1mg/kg、10mg/kgPDTC抑制NF-κB通路活性，减轻SAP大鼠胰腺及肠屏障损伤，以10mg/kg作用更为明显，100mg/kg PDTC不能减轻胰腺及肠屏障损伤，但不论12h或24h100mg/kg PDTC组各指标均未达到SAP组水平。 3RegI、III基因表达可部分通过NF-κB通路调节，肠黏膜损伤或炎症因子刺激致RegI、III表达上调，对黏膜损伤起起作用，其作用机制考虑为多个通路的共同调节，且不除外RegI、III基因之间的存在协同或拮抗作用，对于过度表达的RegI、III是否会加剧SAP时的肠黏膜损伤仍需进一步实验证实。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R576

【参考文献】