Study On Congenital Choledochal Cyst Carcinogenesis Mechanism Via Cyclooxygenase-2 Pathway

Choledochal cyst is a common biliary tract disease in children, sometimes appear clinical manifestations extend to adult. People realized the relationship between choledochal cyst and cholangiocarcinoma gradually that the cholangiocarcinoma incidence rate of choledochal cyst is 3.2%-39.2%, moreover, the incidence rate increased along with the increasing of age since Irwin and Morison reported that choledochal cyst could convert into cholangiocarcinoma. In the last decade, some scholars reported that cholangiocarcinoma also emerged during long-term follow-up even after the cyst was resected completely with hepaticojejunostomy reconstruction. Therefore, the carcinogenic mechanism of choledochal cyst became research focus. Scholars indicated that cancerization of choledochal cyst was a multistage procedure and the blended pancreatic juice and bile refluxing is the initiating agent. At present, the relevant study was mainly focus on proliferation kinetics of bile duct epithelial cells, the mutation of oncogenes and anti-oncogenes et al. But the molecular biologic mechanism of canceration of choledochal cyst is not clarified yet. Recently, some studies reported that COX-2 overexpress in cyst epithelial cells, indicated COX-2 may play carcinogenic role in choledochal cyst.COX enzymes play key roles in the biosynthesis of prostaglandins from arachidonic acid. To date, three isoforms of the COX have been identified, COX-1, COX-2 and COX-3. COX-1 is ubiquitous and produced constitutively in most mammalian tissues to maintain baseline levels of PGE2 to maintain normal physiological function of organism. COX-2 is inducible enzyme, which is normally absent. However, COX-2 is found to be induced by a variety of stimuli such as cytokines, inflammatory cytokines, endotoxin, carcinogens, oncogenes, tumor suppressor genes and so on. Shirvani et al found that when bile salts stimulated Barrett esophagus the COX-2 overexpress which was in the Barrett esophagus. So we speculate that there are possible factors which induce expression of COX-2 in the patients with choledochal cyst.Generally considered, activated COX-2 has two activities. One is COX enzyme which can convert arachidonic acid into PGG2; the other is peroxidase that can convert PGG2 into PGH2 whose main function is to start and promote inflammation reaction. Recent studies show that COX-2 is related closely to the occurrence and development of tumors, especially the tumor which is in the digestive tract, such as esophagus cancer, gastric cancer, colorectal cancer et al. COX-2 not only influence cell cycle and apoptosis through promote expression of apoptosis suppressor genes such as bcl-2 but also affect the angiogenesis of tumors, for example, promote the expression of VEGF. And it can also influence the invasion and metastasis of tumor cells by producing excessive PGs (such as PGE2) to inhibit the function of immune surveillance of organism. So COX-2 may be a possible target which can prevent and therapy tumor effectively, in other words, COX-2 inhibitors may prevent and therapy the occurrence and development of tumors which is confirmed in esophageal carcinoma and colorectal cancer.The aim of this study is to investigate mechanism of the carcinogenesis in choledochal cyst via COX-2 pathway. And propose possible method and strategy which can prevent canceration of choledochal cyst.PARTⅠExpression of cycooxygenase-2, Bcl-2 and Fas protein on the common bile duct epithelia and effect of Bcl-2 and Fas protein on the apoptosis of common bile duct epithelia in rats with incompletely obstructive jaundiceObjective To investigate the expression of cyclooxygenase-2(Cox-2), Bcl-2 and Fas protein on the common bile duct epithelia and the relationship of the expression of Bcl-2 and Fas protein with apoptosis of common bile duct epithelia in rats with incompletely obstructive jaundice. Methods A total of 60 adult male or female Sprague-Dawley (SD) rats were randomly divided into two groups:incompletely obstructive jaundice rats by ligaturing common bile duct (Group A, n= 40) and the sham-operation rats (Group B, n= 20). The two groups of rats were sacrificed at postoperative day 3,7,14 and 21, respectively. Then serum bilirubin levels were tested and common bile duct histopathological changes were observed. Apoptosis of common bile duct tissues was assayed by TUNEL method. The immunohistochemical technique was adopted for detecting the Cox-2, Bcl-2 and Fas protein expression.Results In group A, with the time of incompletely obstructive jaundice prolonging, the value of serum bilirubin increased; apoptotic cells of common bile duct epithelia increased. There was no obvious change in group B. Group A vs group B had significant difference (P < 0.05). In group A, Cox-2 protein weakly positive expression on the 3rd day, positive expression on the 21st day. In group A, Bcl-2 and Fas protein were both weakly positive expression on the 3rd day, positive expression on the 7th day and the 14th day, strongly positive expression on the 21st day. Conclusion Cox-2 and Bcl-2 protein may be relevant with the epithelia proliferation of common bile duct in rats with incompletely obstructive jaundice. It is an important mechanism that the organism maintains itself stable by increasing the amount of apoptotic common bile duct epithelia cells when the common bile duct is incompletely obstructive, Bcl-2 and Fas protein can regulate the apoptosis of common bile duct epithelia in rats with incompletely obstructive jaundice. PART II A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from miceObjective:Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture.Methods:Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts epithelial cells and cultured on rat-tail collagen type I coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10ng/ml epidermal growth factor at 37℃in an incubator with 5% humidified CO2.Results:The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5-6d after culture; they were positive against anticytokeratin-19 immunostaining. And transmission electron microscope showed typical bile duct epithelia with microvilli on the cytomembrance, Golgi complex, massive mitochondria, rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve.Conclusions:This culture technique might be a reliable method for isolation, purification and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the (patho-)physiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.PARTⅢGlycochenodeoxycholate plays a carcinogenic role in murine extrahepatic bile duct epithelial cells via cyclooxygenase-2 pathwayObjective To investigate the effect of bile salt on the proliferation of primary cultured murine extrahepatic bile duct cells (MEBECs) in vitro and to investigate the carcinogenic role of bile salt in the biiiary system base on the COX-2 pathway. And to recommend possible ways and strategies to prevent the canceration of murine extrahepatic bile duct cells.Methods Primary cultured MEBECs were incubated with Glycochenodeoxycholate (GCDC), taurocholate(TC), taurochenodeoxycholate (TCDC), taurodeoxycholate(TDC), and tauroursodeoxycholate (TUDC) at various concentrations(0,50,100,200μmol/L) for 24h. And the cells were incubated with 200μmol/L GCDC containing Nimesulide of various concentrations(0,50,100,200μmol/L) for 24h. The cell proliferation was studied by method of MTT assay. Real-time PCR was applied to measure the expression of cyclooxygenase-2(COX-2) mRNA. Western blot was applied to measure the expression of COX-2 protein. Concentration of prostaglandin (PGE2) was measured by enzyme linked immunosorbent assay (ELISA).Results GCDC at various concentration(50,100,200μmol/L) stimulated proliferation of MEBECs. GCDC also induced COX-2 mRNA expression and PGE2 production and in concentration-dependent manner. In contrast, no marked changes were induced by the other bile salts. Nimesulide at various concentration(50,100,200μmol/L) inhibited the proliferation of MEBECs induced by 200μmol/L GCDC. With the increasing of Nimesulide the expression of COX-2 mRNA and production of PGE2 decreased (P<0.001) induced by 200μmol/L GCDC and in concentration-dependent manner.Conclusions GCDC can induce the proliferation of MEBECs and suggest a carcinogenic potential of GCDC. Nimesulide can inhibit the proliferation of MEBECs induced by GCDC. Nimesulide can also suppress the expression of COX-2, and decease the production of PGE2. Nimesulide may have therapeutic potential against biliary carcinogenesis.