本文選題：黏蛋白-2　切入點：梗阻性黃疸　出處：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：梗阻性黃疸(Obstructive jaundice,OJ)是臨床常見疾病,多由肝內(nèi)或肝外膽管機械性梗阻導(dǎo)致膽汁排出不暢和膽汁淤積所致,其伴隨和誘發(fā)的高膽紅素血癥、內(nèi)毒素血癥、菌血癥及氧化應(yīng)激狀態(tài)的改變等對肝屏障、腸道屏障等機體各臟器功能的影響,一直是近年來關(guān)注的熱點。雖然梗阻性黃疸肝損傷的研究已取得很大進步,但仍有很多損傷機制尚未完全清楚,仍需進一步研究以求更大的突破及發(fā)現(xiàn)�，F(xiàn)代研究逐漸認識到肝內(nèi)膽管在正常情況下能特異性表達黏蛋白(Mucins,MUC),對維持肝屏障的穩(wěn)態(tài)發(fā)揮重要的作用。越來越多的研究發(fā)現(xiàn),肝內(nèi)膽管中MUC的異常表達及分布改變?yōu)槟懙兰膊〉陌l(fā)生發(fā)展及預(yù)后評估發(fā)揮了重要作用。研究發(fā)現(xiàn)肝內(nèi)膽管過度分泌的MUC5和MUC2所致的慢性增生性膽管炎是肝內(nèi)膽管結(jié)石形成的主要致病因素。其中分泌型黏蛋白MUC2是腸黏液層的主要骨架,參與構(gòu)成黏膜的保護屏障。大量的O-型寡聚糖鏈提供了較好的黏附功能,同時相鄰聚糖鏈中不同糖基間通過分子內(nèi)共價鍵連接,促使其彈性凝膠的形成。既往對MUC2的研究主要集中于胃腸道,在正常的腸道黏液層中,MUC2對腸屏障發(fā)揮了至關(guān)重要的保護作用。多項研究證實梗阻性黃疸時腸黏膜屏障損傷,腸道黏液層受損,可導(dǎo)致MUC2蛋白表達改變,腸黏膜因細菌侵襲而發(fā)生穩(wěn)態(tài)失衡,使得炎癥反應(yīng)加重。除此之外,在炎癥性膽管疾病中,肝內(nèi)膽管中MUC2異常表達對肝屏障的破壞作用也同樣引起重視。然而目前尚未發(fā)現(xiàn)有關(guān)OJ大鼠肝內(nèi)膽管中MUC2蛋白及基因表達的研究報道。所以,積極開展OJ大鼠肝屏障損傷與肝內(nèi)膽管中MUC2蛋白及基因表達的實驗研究具有重要臨床意義。目前臨床上對于OJ患者,外科手術(shù)仍是主要治療方法,但術(shù)后并發(fā)癥及死亡率一直居高不下,多數(shù)學(xué)者認為術(shù)前需要解除梗阻,但本身操作也會引起并發(fā)癥,因此目前對于外科手術(shù)前是否需要解除,一直存在爭議。膽汁內(nèi)外引流術(shù)作為臨床上解除膽道梗阻的兩種方法,哪種引流方式更好,迄今尚無統(tǒng)一意見。因此本實驗通過研究膽汁內(nèi)、外引流術(shù)對OJ大鼠肝內(nèi)膽管上皮組織MUC2蛋白及基因表達的影響,為OJ患者臨床治療提供更多指導(dǎo)依據(jù)。具體實驗內(nèi)容如下:目的:探討OJ大鼠肝內(nèi)膽管中MUC2表達與肝屏障損傷的關(guān)系,以及膽汁內(nèi)、外引流術(shù)對OJ大鼠肝內(nèi)膽管中MUC2蛋白及基因表達的影響及意義。方法:將36只體重約200-250g的成年健康雄性SD大鼠隨機分成四組:假手術(shù)組(sham operation,SH),梗阻性黃疸組(obstructive jaundice,OJ),膽汁內(nèi)引流組(internal drainage,ID)及膽汁外引流組(external drainage,ED)。1利用改進的造模方法建立動物模型第一次手術(shù):SH組模型(只做十二指腸牽拉,不結(jié)扎膽管),OJ組模型(分離膽管并結(jié)扎離斷),ID組和ED組模型(操作同OJ組)。第8天行二次手術(shù):SH組模型(只做十二指腸牽拉,不做其他處理),OJ組模型(可見膨大膽管,只做十二指腸牽拉,不做其他處理),ID組模型(可見膨大膽管,引流管一端插入膽管縫線固定,另一端與十二指腸相通并荷包縫合固定),ED組模型(可見膨大膽管,引流管一端插入膽管縫線固定,另一端經(jīng)皮下引至頸背部縫線固定)。2留取標本各組大鼠模型均于2次術(shù)后第7天取材。應(yīng)用去致熱原處理的注射器在門靜脈穿刺抽血1ml用于血清內(nèi)毒素測定;留取下腔靜脈血2ml用于血清肝功能檢測。取肝組織數(shù)塊,HE染色觀察肝臟及肝內(nèi)膽管病理形態(tài)學(xué)變化;應(yīng)用免疫組織化學(xué)染色法(Immunohistochemistry,IHC)檢測肝內(nèi)膽管中MUC2蛋白表達和分布情況;應(yīng)用Real time-PCR檢測肝內(nèi)膽管中MUC2 m RNA的表達水平。結(jié)果:1血清肝功能水平通過肝功能檢測發(fā)現(xiàn)OJ組大鼠血清TBIL(107.440±18.150μmol/L)、ALT(122.390±35.310U/L)、AST(409.760±72.500U/L)較SH組(21.130±3.230μmol/L)、(40.460±9.430U/L)、(89.050±28.840U/L)均明顯升高(*P0.01);ID組大鼠血清中TBIL(30.230±11.860μmol/L)、ALT(56.500±19.190U/L)、AST(178.910±66.870U/L)和ED組大鼠TBIL(35.640±11.860μmol/L)、ALT(59.690±19.700U/L)、AST(109.340±37.900U/L)較OJ組均明顯降低(≠P0.01);ID組與ED組大鼠血清TBIL、ALT之間無顯著差異(P=0.454;P=0.793),然而ED組大鼠血清AST較ID組明顯降低(#P0.05)。2血清內(nèi)毒素水平OJ組(0.940±0.290EU/ml)大鼠血清內(nèi)毒素水平較SH組(0.160±0.100EU/ml)明顯升高(*P0.01);而ID組(0.230±0.120EU/ml)和ED組(0.450±0.180EU/ml)較OJ組血清內(nèi)毒素水平明顯降低(≠P0.01);且ID組較ED組血清內(nèi)毒素水平顯著降低(#P0.05)。3 HE染色結(jié)果SH組:顯示正常的肝小葉結(jié)構(gòu),匯管區(qū)肝內(nèi)膽管排列整齊。OJ組:肝小葉結(jié)構(gòu)消失,匯管區(qū)肝內(nèi)膽管明顯增生且排列紊亂,伴纖維組織增生及大量炎性細胞浸潤。ID、ED組:與OJ組相比肝小葉結(jié)構(gòu)有所恢復(fù),周圍炎性細胞浸潤減輕,但仍有部分纖維組織及肝內(nèi)膽管增生存在。ED組匯管區(qū)纖維組織及肝內(nèi)膽管增生與炎性細胞浸潤程度較ID組明顯。4免疫組化結(jié)果MUC2蛋白在OJ組大鼠增生的肝內(nèi)小膽管中表達明顯增強,陽性顆粒分布較廣泛,且染色較深。而SH組大鼠的肝內(nèi)膽管中MUC2蛋白表達較弱,陽性顆粒密度較小,且顯色強度較弱。OJ組MUC2蛋白表達的平均光密度值(OD 0.022±0.003)較SH組(OD 0.004±0.002)顯著升高,差異具有統(tǒng)計學(xué)意義(*P0.01)。ID組(OD 0.008±0.002)和ED組(OD0.014±0.001)較OJ組均明顯減少(≠P0.01),而ED組MUC2蛋白表達較ID組增強,且差異具有統(tǒng)計學(xué)意義(#P0.05)。5 Real time-PCR結(jié)果OJ組大鼠肝內(nèi)膽管中MUC2 m RNA表達(0.390±0.080)較SH組(0.100±0.030)明顯增高,具有顯著性差異(*P0.01)。ID組(0.170±0.010)和ED組(0.240±0.050)較OJ組均明顯降低(≠P0.01)。然而ED組大鼠肝內(nèi)膽管中MUC2 m RNA表達較ID組增強,差異具有統(tǒng)計學(xué)意義(#P0.05)。結(jié)論:1梗阻性黃疸時,存在嚴重的內(nèi)毒素血癥,肝屏障功能嚴重受損,組織學(xué)上,大鼠的肝內(nèi)膽管明顯增生且排列紊亂,并伴纖維組織增生及大量炎性細胞浸潤,在肝內(nèi)的小膽管中MUC2蛋白可表達增強,提示MUC2可能參與了梗阻性黃疸大鼠肝屏障損傷的發(fā)病過程。2通過膽汁內(nèi)、外引流術(shù)均可明顯減輕內(nèi)毒素血癥,并不同程度改善肝屏障功能,組織學(xué)上,膽汁引流后的大鼠仍有部分纖維組織及肝內(nèi)膽管增生存在,其周圍炎性細胞浸潤明顯減輕,同時肝內(nèi)小膽管中MUC2蛋白及基因表達受到明顯抑制,且相對于膽汁外引流術(shù),膽汁內(nèi)引流術(shù)可更大程度逆轉(zhuǎn)這種病理改變,提示膽汁內(nèi)引流術(shù)優(yōu)于外引流術(shù)的機制可能與肝內(nèi)膽管中MUC2蛋白及基因表達調(diào)控有關(guān)。
[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)媯，

本文編號：1669263