【作者】 于洪海；

【导师】 冯志杰；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 急性胰腺炎(acute pancreatitis, AP)是临床上常见的急腹症之一,常常并发肝、肾、肺、肠道等胰外器官损伤。AP肝损伤的机制仍未完全阐明,目前的研究认为与炎性细胞因子、肝细胞凋亡、氧化应激、微循环障碍等多种因素有关,是一个复杂的病理生理过程。P物质(substance P)是神经肽中速激肽家族的重要成员,广泛分布于中枢神经系统及周围神经系统,具有痛觉传导、神经源性炎症、内脏平滑肌收缩及血管扩张等许多生物学活性。P物质在调节炎性细胞的游走中具有重要作用,可引起中性粒细胞的趋化以及白细胞介素8(IL-8)的合成与释放,同时还可引起肥大细胞释放组胺,从而促成炎症反应发生,是神经纤维与炎性细胞间相互作用的介质。P物质与神经激肽受体1(neurokinin receptor, NK-1R)亲合力最强,因而NK-1R又称为P物质受体。NK-1R在神经细胞、血管内皮细胞、成纤维细胞、以及炎性细胞(如淋巴细胞、单核巨噬细胞、中性粒细胞)的细胞膜上表达。P物质与NK-1R结合后,可以抑制胰腺外分泌,减轻各种消化酶对胰腺及胰周组织的损害。但同时还引起血管扩张、白细胞黏附血管壁并游出血管外、血浆外渗等反应。P物质引起的反应实质上是机体对外界有害刺激的防御反应,但过分剧烈则产生不良后果。AP患者肝功能异常率高,肝功能的损害程度与AP病情呈正相关,而一旦发生肝衰竭则预后不良。研究P物质在急性胰腺炎肝损伤时的作用对揭示AP肝损伤机制和提供治疗思路具有一定的意义。目的:研究通过建立大鼠AP模型,并给予NK-1R拮抗剂L-703,606干预,观察肝脏中P物质、NK-1R的表达以及肝细胞凋亡的情况,探讨P物质在AP肝损伤中的作用,探讨NK-1R拮抗剂对AP肝损伤的治疗作用。方法:1动物模型的制备:选取Sprague-Dawley(SD)雄性大鼠,以10%水合氯醛腹腔注射麻醉,取上腹正中作切口进入腹腔,以4号手术线结扎近肝门端胰胆管,然后用4号加工钝头头皮静脉针于十二指肠乳头附近逆行插入胰胆管,以0.2ml/min速度注入5%牛磺胆酸钠(0.1ml/100g),推药后以左手拇指按紧胰胆管入十二指肠处,观察3min,待胰腺出现明显充血后,剪去结扎线,关腹。2实验分组:将雄性SD大鼠54只,随机分为假手术组(SO group, n=18)、AP模型组(AP group, n=18)、AP+L-703,606组(AP+L-703,606 group, n=18),后以不同时间点3h、6h、12h分为3个亚组。其中AP+L-703,606组于造模前15min腹腔内注射L-703,606(250nmol/kg)。3生化指标测定:取血清样本,采用全自动血清生化分析仪检测血清淀粉酶、脂肪酶、谷丙转氨酶(alanine aminotransferase,ALT)由河北医科大学第二医院生化室协助测定。4肝脏和胰腺组织学检查:采用石蜡切片,HE染色。5肝脏P物质和NK-1R的免疫组织化学染色:石蜡切片脱蜡至水,抗原热修复,3%过氧化氢孵育15min,山羊血清封闭30min,滴加一抗4℃过夜。阴性对照用PBS代替一抗。先后滴加二抗、三抗各15min,后用3,3-二氨基苯联胺(DAB)显色,苏木素复染后封片。以细胞浆内呈现黄褐色颗粒为阳性表达。6凋亡测定:采用TUNEL检测试剂盒检测肝细胞的凋亡。结果:1大鼠胰腺的病理学变化:肉眼和光镜所见SO组胰腺及周围组织结构始终大致正常;AP组和AP+L-703,606组,胰腺及周围组织水肿、出血、坏死,且随时间延长损伤程度逐渐加重,腹水和中性粒细胞也逐渐增多;2大鼠肝脏的病理学变化:肉眼和光镜所见SO组肝脏组织结构始终正常;AP组和AP+L-703,606组随时间推移,肝细胞肿胀、变性、坏死和肝索结构解离破坏的范围逐渐增大,程度逐渐加重,炎性细胞浸润也增多。但AP+L-703,606组较AP组肝脏组织的损伤程度较轻。3血清淀粉酶的变化:在各时间点,SO组无明显变化,AP组和AP+L-703,606组与SO组比较均显著升高(P<0.01)。AP组和AP+L-703,606组间无差异。4血清脂肪酶的变化:在各时间点,SO组无明显变化,AP组和AP+L-703,606组与SO组比较均显著升高(P<0.05)。AP组和AP+L-703,606组间无差异。5血清谷丙转氨酶的变化:各时间点,SO组无明显变化,AP组和AP+L-703,606组与SO组比较均显著升高(P<0.05)。AP组和AP+L-703,606组间无差异,但AP+L-703,606组各时间点均低于AP组。6肝脏P物质变达的变化:SO组,有散在分布的P物质表达于肝细胞胞浆内,成棕黄色颗粒。AP组和AP+L-703,606组,均有较强的表达。免疫组化评分显示AP+L-703,606组表达强度低于AP组。7肝脏NK-1R表达的变化:SO组,NK-1R表达于肝细胞胞浆,染色为棕黄色颗粒。AP组和AP+L-703,606组表达均强于SO组。免疫组化评分显示AP+L-703,606组表达较AP组弱。8肝细胞凋亡的变化:SO组几乎没有阳性细胞表达,AP组和AP+L-703,606组均可见细胞核呈棕黄色染色的阳性细胞,且随时间延长数量增加。但AP+L-703,606组数量少于AP组。结论:1逆行胆胰管内注射5%牛磺胆酸钠,血清淀粉酶、脂肪酶升高,胰腺组织HE染色观察到胰腺及周围组织水肿、出血和坏死等表现,证实大鼠AP模型制备成功。2 AP组和AP+L-703,606组大鼠血清ALT均明显升高,HE染色观察到肝脏组织结构破坏,肝细胞变性、坏死,TUNEL检测到明显的肝细胞凋亡,说明AP时大鼠肝脏出现损伤。3 AP大鼠肝脏组织中P物质及其受体NK-1R表达增加,表明P物质与AP肝损伤有密切联系。4 L-703,606是NK-1R的特异性拮抗剂,它通过阻断P物质与NK-1R结合抑制P物质发挥生物学效应。给予L-703,606后P物质和NK-1R表达降低,血清ALT降低,肝细胞凋亡减少,肝脏组织损伤减轻。因此认为,AP时P物质通过与NK-1R结合参与了肝损伤,是AP肝损伤的重要原因之一。更多还原

【Abstract】 Acute pancreatitis(AP) is a frequent acute abdomen in clinic, causes damages not only to pancreas, but also to distant organs, such as liver, kidney, lung, intestines etc. The mechanisms of hepatic injury during AP are not clear yet. The existing studys shows that, hepatic injury is a complicatide pathology process with many facters such as inflammatory cytokines, hepatic apoptosis, oxidative stress, microcirculation disturbance etc.Substance P is an important member of tachykinin family which belong to neuropeptide. It is generally distributed in central nervous system and peripheral nerveous system. Substance P has many biological activities, such as algesia conduction, neurogenic inflammation, visceral smooth muscle contraction and angiectasis etc. It has important effect in regulating inflammatory cells transmigration. It is a medium between nerve fiber and inflammatory cell, becaues it can promote inflammatory reaction happening by neutrophil aggregation, interleukin 8 synthesis and release, and mastocyte release histamine. Substance P has the strongest avidity with neurokinin receptor-1(NK-1R). So NK-1R also called substance P receptor. NK-1R express at cell envelope of nerve cell, vascular endothelial cell, fibroblast and inflammatory cell (such as lymphocyte, mononuclear macrophage , neutrophil). As substance P combine with NK-1R, it can inhibit exocrine pancreas, lighten pancreas and peripancreatic tissue damage caused by various digestive enzymes. But it also causes angiectasis, blood plasma exosmose, white blood cell adhesion and emigration out of blood vessel. The reaction caused by substance P is a defensive reaction of organsim to external destructive stimulus virtually, but it can leads to harmful result if it over servere. Patients with AP have a high rate of hepatic disfunction, and hepatic injury level is direct correlation with AP condition. It means unfavourable prognosis, when hepatic failure happens. Therefor, it is important to study the effect of substance P in AP hepatic injury. And it may helps to reveal the mechanism of AP hepatic injury and provide an idea for treatment.Objective:To investigate expressions of substance P, NK-1R and hepatic apoptosis by establishing AP model and administrating L-703,606 which is an antagon of NK-1R, to explore the effect of substance P in hepatic injury in AP and the therapeutical effect of NK-1R antagon.Method:1 Animal mode: The experiment was performed in SD rats under anesthesia injected intra-peritoneally with 10% chloral hydrate. Then, the abdomen median incision was performed. Use #4 ligature to ligate bile duct near hepatic portal before bile duct was injected antidromicly by microinjector with 5% Sodium Cholate (0.1ml/100g) at the speed of 0.2ml/min. Then press bile duct entering duodenum by thumb of left hand. 3 minutes later, when pancrea appears obviously hyperemia, cut off ligature and suture abdomen.2 Animal groups: All SD rats (n=54) were divided into 3 groups at random: sham-operation group (n=18), AP group (n=18) and AP+L-703,606 group (n=18). Then every group was divided into 3 subgroups (n=6) at different time points: 3h、6h、12h. The rats in AP+L-703,606 group were injected intra-peritoneally with L-703,606(250nmol/kg) 15 minutes before injected Sodium Cholate.3 Mensuration of biochemical parameters: The levels of serum amylase, lipase, alanine aminotransferase were determined respectively by automatic biochemical analyzer.4 Pancreatic gland and liver histological analysis: Paraffin section was stained by hematoxylin-eosin (HE).5 Immunohistochemical staining of substance P and NK-1R: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 15min. The sections were dripped with goat serum for 30min, then incubated with first antibody at 4℃overnight. In negative control group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 15min respectively. The results were visualized by using 3,3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin.Hepatic apoptosis: Using TUNEL detect and measurement kit to detect apoptotic hepatocyte.Results:1 Histological findings of pancrea: It was seen by naked eyes and light microscope that the pancreatic gland and its surrounding tissue of sham-operation group was approximatly normal; areas and degrees of dropsy, hemorrhage and necrosis of pancreatic gland and its surrounding tissue were being more and more severe, and the seroperitoneum and and neutrophil were growing in AP group and AP+L-703,606 group.2 Histological findings of hepatic: It was seen by naked eyes and light microscope that the liver tissue of sham-operation group was approximatly normal; areas and degrees of cellular swelling, denaturation, necrosis and hepatic cord structure decollement were being more and more severe, and inflammatory cell infiltrate were growing in in AP group and AP+L-703,606 group. Compared with AP group the hepatic injury was lighter in AP+L-703,606 group.3 Serum amylase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum amylas were increased in AP group and AP+L-703,606 group (P<0.05). But there was no significant difference between AP group and AP+L-703,606 group. 4 Serum lipase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum lipase were increased in AP group and AP+L-703,606 group (P<0.05). But there was no significant difference between AP group and AP+L-703,606 group.5 Serum alanine aminotransferase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum alanine aminotransferase were increased in AP group and AP+L-703,606 group (P<0.05). There was no significant difference between AP group and AP+L-703,606 group, but AP+L-703,606 group had lower level than AP group at each time point.6 Expression of substance P: In sham-operation group, substance P was a little expressed in intracytoplasm of hepatocyte cells. We found an overexpression of substance P in AP group and AP+L-703,606 group. Immunohistochemistry score showed that substance P expressed in AP+L-703,606 group was lower than in AP group.7 Expression of NK-1R: In sham-operation group, NK-1R was expressed a little in intracytoplasm of hepatocyte cells. We found an overexpression of NK-1R in AP group and AP+L-703,606 group. Immunohistochemistry score showed that substance P expressed in AP+L-703,606 group was lower than in AP group. 8 Hepatic apoptosis: There were hardly any apoptotic cells in sham-operation group. In AP group and AP+L-703,606 group, we can see many apoptotic cells tincted buffy and increased by the time. But AP+L-703,606 group had less amount than AP group.Conclusions:1 On the basis of histological findings and increase of serum amylase and lipase from this study, we concluded that AP model group has been successfully manufactured by bile duct inject antidromic with 5% Sodium Cholate.2 On the basis of histological findings ,increase of serum alanine aminotransferase and hepatic apoptosis from this study, we concluded that AP rats have apparente hepatic injury.3 The overexpression of substance P and NK-1R in AP rats shows substance P play role in hepatic injury.4 L-703,606 is an specific antagon of NK-1R. It play biological effect by block NK-1R to combine with substance P. It can decrease serum alanine aminotransferase, hepatocyte apoptosis, expression of substance P and NK-1R, ameliorate liver tissue damage when give to acute pancreatitic rats. We suppose that substance P play an important role in hepatic injury by combined to NK-1R during acute pancreatitis.更多还原