Expression And Significance Of SHH、E-cadherin And IL-6Expression In Human Chronic Calculous Cholecystitis

Background and Objective:Sonic Hedgehog (SHH) signal plays an important role in the regulation of early embryonic development and organ differentiation process. E-cadherin as a single transmembrane glycoprotein widely distributed in embryos and mature epithelial, maintaining the integrity of the organizational structure form and cell polarity in physiological conditions. Interleukin6is an important component of the cytokine network, which involve in the inflammatory response and immunity function. Recent research shows that SHH signaling pathway, E-cadherin and IL-6participate in tumors development progress, but the roles of SHH signaling pathway, E-cadherin and IL-6in inflammatory disease, especially in chronic calculous cholecystitis are not clear. Further study of the mechanism from chronic calculous cholecystitis to gallbladder cancer is expected to find specific molecular markers for early diagnosis and targeted therapy.Methods:1. Experimental design:120cases of postoperative tissue samples of well-preserved chronic calculous cholecystitis from Wuhan General Hospital of Guangzhou Military were collected from January2005to December2010, which included48cases of male,72cases of female, with an average age of51.8years. According to the classification standards of WangyangKun etc:45patients with mild disease,45cases with moderate disease,30cases with severe disease. The control group was tissue samples from36cases of gallbladder adenocarcinoma patients, including20cases male,16female, with an average age of61.4years, all the patients were not given radiotherapy or chemotherapy before surgery. Mild:clinical course<1year; B Ultrasound examination show mild gallbladder wall thickening or have no obvious nodular thickening; Histopathological examination reveal mucosal epithelium remain intact. Glandular epithelium cells show no atypia, the main infiltrating inflammatory cells are lymphocyte, with or without Rokitansky-Aschoff sinuses; mild glandular hyperplasia, mucous gland enhance. Moderate:inflammation between mild and severe group. Severe:clinical course>5years; B ultrasound examination show gallbladder atrophy or enlargement, with diffuse or limitations (nodular) gallbladder wall thickening, many accompanied by polypoid lesion. Histopathological examination reveals that mucosal epithelium part or most fall off. The gland hyperplasia with irregular dilation, Glandular epithelium cells active hyperplasia. The inflammatory cell infiltrated seriously, and the lymphoid follicles-like structure are formed. The fibrous connective tissues are dysplasia and most with Rokitansky-Aschoff sinuses, mucous gland enhance.2. HE experimental method:Paraffin wax specimen were sliced with4μm thickness, the slice contained gallbladder mucous epithelial, submucosal interstitium, muscle layer. HE dyeing and immunohistochemical staining were performed. HE dyeing:line gradient wax dehydration and hydration, sappanwood stained5min, then eosin dyed2min, conventional dehydration, transparency, sealing piece.3. Immunohistochemical experimental method:After gradient wax dehydration and hydration, the slice antigen was repaired by heat; then cooled to room temperature after washed5min, and3%hydrogen peroxide incubation to blocked the endogenous peroxidase; Phosphate buffer (PBS) washed2min with a total of three times; Add nonimmunologic animal serum to blocked nonspecific binding,4℃moisturizing, overnight; PBS washed5min, a total of three times, added second antibody, kept in room temperature for30min; PBS washed2min, a total of three times, and then stained with DAB, water flushed for5min, sappanwood restained, step-down dehydration, transparency, and then sealed with neutral gum.4. Immunohistochemical image analysis’, immune complex deposition and immune reaction intensity were observed under optical-microscopy. Positive judgment standard:when the cell membrane and (or) cytoplasm appeared brown, then it was judged as positive cell. Observation parameters:positive expression of SHH, E-cadherin and IL-6in the tissues of chronic calculous cholecystitis and gallbladder cancer--average intensity of gray value, positive unit. Image analysis and statistics analysis:chose5fields on each slide under400times magnification. Use HMIAS-2000high-resolution colourful pathological report analysis system of Wuhan Qianping company to determined the average gray intensity and positive unit value.5. Data analysis: Statistical software SPSS13.0was used for random designed analysis of variance (One-Way ANOVA) and Pearson related analysis, all of the data was expressed with x±s, P<0.05for difference with a statistical significance.Results:1. The correlation of SHH expression and the clinical pathological characters: SHH expressed mainly in the gallbladder mucosal epithelium and gallbladder cancer cell membranes and the cytoplasms. SHH expression in chronic mild, moderate, severe calculous cholecystitis and control group (gallbladder cancer group) tissue samples were statistically significant (P<0.01). Along with the different pathological damage degree of chronic calculous cholecystitis from light to heavy, the positive particles of the average gray and positive unit value were significant rising and the control group (gallbladder cancer group) was highest. SHH protein expression had no relation with gender and age (P>0.05), SHH protein expression and stone size (≥3cm in diameter or<3cm in diameter) were statistically significant (P<0.01).2. The correlation of E-cadherin expression and the clinical pathological characters:E-cadherin expressed mainly in the gallbladder mucosal epithelium and gallbladder cancer cell membranes and the cytoplasms. E-cadherin expression in chronic mild, moderate, severe calculous cholecystitis and control group (gallbladder cancer group) tissue samples were statistically significant (P<0.01). Along with the different pathological damage degree of chronic calculous cholecystitis from light to heavy, the positive particles of the average gray and positive unit value were significant decreasing and the control group (gallbladder group) was lowest. E-cadherin protein expression had no relation with gender and age (P>0.05), E-cadherin protein expression and stone size (>3cm in diameter or<3cm in diameter) were statistically significant (P<0.01).3. The correlation of IL-6expression and the clinical pathological characters: IL-6expressed mainly in the gallbladder mucosal epithelium, submucosal interstitial and gallbladder cancer cell cytoplasm. IL-6expression in chronic mild, moderate, severe calculous cholecystitis and control group (gallbladder cancr group) tissue samples were statistically significant (P<0.01). Along with the different pathological damage degree of chronic calculous cholecystitis from light to heavy, the positive particles of the average gray and positive unit value were significant rising and the control group (gallbladder cancer group) was highest. IL-6protein expression had no relation with gender and age (P>0.05), SHH protein expression and stone size (>3cm in diameter or<3cm in diameter) were statistically significant (P<0.01).4. The correlation analysis of SHH, E-cadherin and IL-6protein expressions in chronic calculous cholecystitis tissue:the paired data Pearson relevant analysis showed that SHH expression and E-cadherin expression were inversely related (r average gray=-0.71, r positive unit=-0.68, P<0.01), IL-6expression and E-cadherin expression were inversely related (r average gray=-0.75, r positive unit= -0.83, P<0.01), SHH expression and IL-6expression were positively related (r average gray=0.71, r positive unit=0.88, P<0.01).Conclusions:The results of this study showed that SHH, E-cadherin and IL-6expressions correlated to the severity of chronic calculous cholecystitis and the size of gallbladder stone. But they had no relationship with age, gender and pathological factors. The Pearson related analysis found that SHH and IL-6expressions had positive correlation, but SHH, IL-6and E-cadherin expressions were inversely related respectively. Our results suggested that, in chronic calculous cholecystitis, when the gallbladder inflammation was aggravating, gallbladder mucosal epithelium was damaged, along with cell polarity lost and IL-6released increase. Overexpression of SHH signaling pathway could induce gallbladder epithelial cell excessive proliferation and progression. As a suppressor gene, downregulation of E-cadherin could cause gallbladder epithelial cells destruction and lost of polarity, and SHH signaling pathway had some correlations with E-cadherin, both of them participated in the progression of gallbladder cancer. Some studies showed that downregulation of E-cadherin activated SHH signaling pathway, the transcriptional factors Glil of SHH pathway could activated the E-cadherin inhibitor,Snail, to mediated epithelial cells skeleton damage. So we speculated that, as the main reason of chronic cholecystitis, cystic calculous induced long-term mechanical damage of gallbladder mucosal epithelium, and polarity damaged, inflammatory cell infiltration, IL-6released increase. Mucosal epithelium skeleton destruction, lost of polarity and downregulation of E-cadherin could then activated SHH signaling pathway, and induced over proliferation of gallbladder epithelial cells, at the same time, the activation of SHH signaling pathway through the activation of Snail could inhibited E-cadherin expression, thus made the epithelial cells skeleton destructed further, as the same with lost of polarity, which eventually led to tumor. This study suggests that, in the process of chronic calculous cholecystitis, long-term inflammation stimulate the gallbladder epithelial cells skeleton destruction, loss of polarity, and then induce IL-6lose control and E-cadherin downregulation or deletion, which activate SHH signaling pathway. They all participate in the development of gallbladder cancer. The mechanism of the occurrence of gallbladder cancer provides new theory basis for future development of molecular target therapy.