胆汁内、外引流术对梗阻性黄疸大鼠肝内胆管中MUC2蛋白及基因表达的影响

发布时间：2018-03-26 19:24

  本文选题：黏蛋白-2　切入点：梗阻性黄疸　出处：《河北医科大学》2017年硕士论文

【摘要】：梗阻性黄疸(Obstructive jaundice,OJ)是临床常见疾病,多由肝内或肝外胆管机械性梗阻导致胆汁排出不畅和胆汁淤积所致,其伴随和诱发的高胆红素血症、内毒素血症、菌血症及氧化应激状态的改变等对肝屏障、肠道屏障等机体各脏器功能的影响,一直是近年来关注的热点。虽然梗阻性黄疸肝损伤的研究已取得很大进步,但仍有很多损伤机制尚未完全清楚,仍需进一步研究以求更大的突破及发现。现代研究逐渐认识到肝内胆管在正常情况下能特异性表达黏蛋白(Mucins,MUC),对维持肝屏障的稳态发挥重要的作用。越来越多的研究发现,肝内胆管中MUC的异常表达及分布改变为胆道疾病的发生发展及预后评估发挥了重要作用。研究发现肝内胆管过度分泌的MUC5和MUC2所致的慢性增生性胆管炎是肝内胆管结石形成的主要致病因素。其中分泌型黏蛋白MUC2是肠黏液层的主要骨架,参与构成黏膜的保护屏障。大量的O-型寡聚糖链提供了较好的黏附功能,同时相邻聚糖链中不同糖基间通过分子内共价键连接,促使其弹性凝胶的形成。既往对MUC2的研究主要集中于胃肠道,在正常的肠道黏液层中,MUC2对肠屏障发挥了至关重要的保护作用。多项研究证实梗阻性黄疸时肠黏膜屏障损伤,肠道黏液层受损,可导致MUC2蛋白表达改变,肠黏膜因细菌侵袭而发生稳态失衡,使得炎症反应加重。除此之外,在炎症性胆管疾病中,肝内胆管中MUC2异常表达对肝屏障的破坏作用也同样引起重视。然而目前尚未发现有关OJ大鼠肝内胆管中MUC2蛋白及基因表达的研究报道。所以,积极开展OJ大鼠肝屏障损伤与肝内胆管中MUC2蛋白及基因表达的实验研究具有重要临床意义。目前临床上对于OJ患者,外科手术仍是主要治疗方法,但术后并发症及死亡率一直居高不下,多数学者认为术前需要解除梗阻,但本身操作也会引起并发症,因此目前对于外科手术前是否需要解除,一直存在争议。胆汁内外引流术作为临床上解除胆道梗阻的两种方法,哪种引流方式更好,迄今尚无统一意见。因此本实验通过研究胆汁内、外引流术对OJ大鼠肝内胆管上皮组织MUC2蛋白及基因表达的影响,为OJ患者临床治疗提供更多指导依据。具体实验内容如下:目的:探讨OJ大鼠肝内胆管中MUC2表达与肝屏障损伤的关系,以及胆汁内、外引流术对OJ大鼠肝内胆管中MUC2蛋白及基因表达的影响及意义。方法:将36只体重约200-250g的成年健康雄性SD大鼠随机分成四组:假手术组(sham operation,SH),梗阻性黄疸组(obstructive jaundice,OJ),胆汁内引流组(internal drainage,ID)及胆汁外引流组(external drainage,ED)。1利用改进的造模方法建立动物模型第一次手术:SH组模型(只做十二指肠牵拉,不结扎胆管),OJ组模型(分离胆管并结扎离断),ID组和ED组模型(操作同OJ组)。第8天行二次手术:SH组模型(只做十二指肠牵拉,不做其他处理),OJ组模型(可见膨大胆管,只做十二指肠牵拉,不做其他处理),ID组模型(可见膨大胆管,引流管一端插入胆管缝线固定,另一端与十二指肠相通并荷包缝合固定),ED组模型(可见膨大胆管,引流管一端插入胆管缝线固定,另一端经皮下引至颈背部缝线固定)。2留取标本各组大鼠模型均于2次术后第7天取材。应用去致热原处理的注射器在门静脉穿刺抽血1ml用于血清内毒素测定;留取下腔静脉血2ml用于血清肝功能检测。取肝组织数块,HE染色观察肝脏及肝内胆管病理形态学变化;应用免疫组织化学染色法(Immunohistochemistry,IHC)检测肝内胆管中MUC2蛋白表达和分布情况;应用Real time-PCR检测肝内胆管中MUC2 m RNA的表达水平。结果:1血清肝功能水平通过肝功能检测发现OJ组大鼠血清TBIL(107.440±18.150μmol/L)、ALT(122.390±35.310U/L)、AST(409.760±72.500U/L)较SH组(21.130±3.230μmol/L)、(40.460±9.430U/L)、(89.050±28.840U/L)均明显升高(*P0.01);ID组大鼠血清中TBIL(30.230±11.860μmol/L)、ALT(56.500±19.190U/L)、AST(178.910±66.870U/L)和ED组大鼠TBIL(35.640±11.860μmol/L)、ALT(59.690±19.700U/L)、AST(109.340±37.900U/L)较OJ组均明显降低(≠P0.01);ID组与ED组大鼠血清TBIL、ALT之间无显著差异(P=0.454;P=0.793),然而ED组大鼠血清AST较ID组明显降低(#P0.05)。2血清内毒素水平OJ组(0.940±0.290EU/ml)大鼠血清内毒素水平较SH组(0.160±0.100EU/ml)明显升高(*P0.01);而ID组(0.230±0.120EU/ml)和ED组(0.450±0.180EU/ml)较OJ组血清内毒素水平明显降低(≠P0.01);且ID组较ED组血清内毒素水平显著降低(#P0.05)。3 HE染色结果SH组:显示正常的肝小叶结构,汇管区肝内胆管排列整齐。OJ组:肝小叶结构消失,汇管区肝内胆管明显增生且排列紊乱,伴纤维组织增生及大量炎性细胞浸润。ID、ED组:与OJ组相比肝小叶结构有所恢复,周围炎性细胞浸润减轻,但仍有部分纤维组织及肝内胆管增生存在。ED组汇管区纤维组织及肝内胆管增生与炎性细胞浸润程度较ID组明显。4免疫组化结果MUC2蛋白在OJ组大鼠增生的肝内小胆管中表达明显增强,阳性颗粒分布较广泛,且染色较深。而SH组大鼠的肝内胆管中MUC2蛋白表达较弱,阳性颗粒密度较小,且显色强度较弱。OJ组MUC2蛋白表达的平均光密度值(OD 0.022±0.003)较SH组(OD 0.004±0.002)显著升高,差异具有统计学意义(*P0.01)。ID组(OD 0.008±0.002)和ED组(OD0.014±0.001)较OJ组均明显减少(≠P0.01),而ED组MUC2蛋白表达较ID组增强,且差异具有统计学意义(#P0.05)。5 Real time-PCR结果OJ组大鼠肝内胆管中MUC2 m RNA表达(0.390±0.080)较SH组(0.100±0.030)明显增高,具有显著性差异(*P0.01)。ID组(0.170±0.010)和ED组(0.240±0.050)较OJ组均明显降低(≠P0.01)。然而ED组大鼠肝内胆管中MUC2 m RNA表达较ID组增强,差异具有统计学意义(#P0.05)。结论:1梗阻性黄疸时,存在严重的内毒素血症,肝屏障功能严重受损,组织学上,大鼠的肝内胆管明显增生且排列紊乱,并伴纤维组织增生及大量炎性细胞浸润,在肝内的小胆管中MUC2蛋白可表达增强,提示MUC2可能参与了梗阻性黄疸大鼠肝屏障损伤的发病过程。2通过胆汁内、外引流术均可明显减轻内毒素血症,并不同程度改善肝屏障功能,组织学上,胆汁引流后的大鼠仍有部分纤维组织及肝内胆管增生存在,其周围炎性细胞浸润明显减轻,同时肝内小胆管中MUC2蛋白及基因表达受到明显抑制,且相对于胆汁外引流术,胆汁内引流术可更大程度逆转这种病理改变,提示胆汁内引流术优于外引流术的机制可能与肝内胆管中MUC2蛋白及基因表达调控有关。
[Abstract]:Obstructive jaundice (Obstructive jaundice OJ) is a common clinical disease, from intrahepatic or extrahepatic bile duct obstruction leads to poor discharge of bile and cholestasis caused by the associated and induced hyperbilirubinemia, endotoxemia, bacteremia and oxidative stress change on liver barrier effect of intestinal barrier and other body organs the function, has been a hot spot of attention in recent years. Although the study of obstructive jaundice liver injury has made great progress, but there are still a lot of damage mechanisms are not completely understood, and further research is needed for greater breakthrough and discovery. Modern research gradually realized the intrahepatic bile duct specific expression in normal circumstances (Mucins, MUC), play an important role in the homeostasis of liver barrier. More and more studies have found that abnormal expression and distribution of MUC in the intrahepatic bile duct changes of biliary tract diseases. Development and prognosis evaluation plays an important role. The study found that chronic proliferative cholangitis of intrahepatic bile duct and excessive secretion of MUC5 induced by MUC2 is the main pathogenic factor in intrahepatic calculi. The secretory mucin MUC2 is the main skeleton of intestinal mucus layer, a protective barrier in the mucosa. A large number of O- type oligosaccharides the chain offers good adhesion, while adjacent chitosan chain of different glycosylation through intramolecular covalent bond formation, the elastic gel. The research on MUC2 history mainly concentrated in the gastrointestinal tract, intestinal mucus layer in normal, MUC2 plays a crucial role in protecting intestinal barrier. A number of studies have confirmed obstructive jaundice, intestinal mucosal barrier injury, intestinal mucous layer is damaged, can cause changes in the expression of MUC2 protein, the intestinal mucosa due to bacterial invasion and homeostasis, which aggravates inflammatory reactions in addition. In inflammatory diseases, bile duct, intrahepatic bile duct in the abnormal expression of MUC2 on liver damage barrier also paid attention to. However, has not yet found the reports on the expression of MUC2 protein and OJ gene in intrahepatic bile duct in rats. Therefore, it has important clinical significance to actively carry out the experimental study on the expression of MUC2 protein and gene in liver barrier liver injury and OJ bile duct in rats. The current clinical for OJ patients, surgery is the main treatment method, but the postoperative complications and mortality rate has been high, the majority of scholars believe that the need to remove the obstruction before operation, but the operation will cause complications, therefore at present whether to release before surgery, there has been internal and external biliary drainage as controversial. Two methods of clinical biliary obstruction, which means better drainage, so far there is no unified opinion. Therefore this experiment through the research of bile Within the drainage effect on the expression of MUC2 gene and protein in bile duct epithelial tissue of OJ rat liver, to provide more guidance for clinical treatment of patients with OJ. The experimental results are as follows: Objective: To investigate the relationship between MUC2 expression and liver injury barrier in OJ rats in the intrahepatic bile duct and bile drainage, and its significance effect of OJ on the expression of MUC2 in intrahepatic bile duct in rats in protein and gene. Methods: 36 weight of 200-250g healthy adult male SD rats were randomly divided into four groups: sham operation group (sham operation, SH), obstructive jaundice group (obstructive, jaundice, OJ), internal biliary drainage group (internal drainage, ID) and external biliary drainage group (external drainage, ED) improved modeling method to establish the first surgery animal model using.1: SH model group (only duodenal stretch, no bile duct ligation), OJ model group (and transection of the bile duct separation ligation), ID group and ED group (model The operation with the OJ group). The two operation eighth days: SH model group (only duodenal stretch, no other treatment), group OJ (visible swelling bold tube, only the duodenal stretch, no other treatment), group ID (visible swelling bold tube, drainage tube end is inserted into the common bile duct suture fixation, and the other end is communicated and duodenal purse string suture), group ED (visible swelling bold tube, drainage tube end is inserted into the bile duct suture fixation, the other end to the back of the neck subcutaneous suture fixation).2 specimens from model rats were 2 after seventh days. The application by syringe the pyrogen in the portal vein puncture for blood 1ml serum endotoxin determination; collected inferior vena cava blood 2ml for detection of liver function. The liver tissue fragments, observe the changes of liver and intrahepatic bile duct pathological HE staining; using immunohistochemical staining method (Immunohistochemistry, IHC)妫，

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