[Abstract] Objective To investigate survivin and Smac role and significance in the development of the extrahepatic bile duct. Methods 27 cases of extrahepatic bile duct cancer, 10 cases of dysplasia, and 5 cases of normal epithelial tissue underwent immunohistochemical staining Survivin and Smac expression. The results of the semi-quantitative method evaluation, analysis and comparison of statistical methods. Results The positive expression rate of Survivin normal epithelial tissue, extrahepatic bile duct dysplasia and cancer tissue were 20%, 50% and 74.1%, respectively, Survivin expression in normal bile duct epithelial tissue and cancer tissue difference was statistically significant (P = 0.037 ). Smac normal epithelial tissue in the extrahepatic bile duct dysplasia and cancer tissue positive expression rate of 40%, 50% and 85.2%, dysplasia and cancer tissue expression difference was statistically significant (P = 0.041). Survivin and Smac expression with extrahepatic cholangiocarcinoma histopathological characteristics and clinical stage. Conclusion Survivin and Smac apoptosis-related genes involved in the development of extrahepatic cholangiocarcinoma, may play an important role in the process of malignant transformation further cancer from normal epithelial tissue to dysplasia.
Key words dysplasia Survivin Smac immunohistochemistry extrahepatic cholangiocarcinoma
Extrahepatic bile duct cancer is a relatively rare disease, slow growth covert, difficult to early detection, often as the first symptom of obstructive jaundice, low rate of surgical resection, the prognosis is poor. Tumor development is closely related to apoptosis, previous studies have shown that, in extrahepatic cholangiocarcinoma happened carcinoma sequence, apoptosis-related genes Bcl 2 and Bax gradually increased . Survivin and Smac is a newly discovered protein of apoptosis-related genes and their expression in extrahepatic bile duct cancer and precancerous lesions has not been reported. In this study, using immunohistochemical staining Survivin and Smac expression in of extrahepatic cholangiocarcinoma its profiled organization, to investigate the of extrahepatic cholangiocarcinoma incidence of the development and clinical prognosis relations.
1 Materials and Methods
1.1 specimen collection
Extrahepatic cholangiocarcinoma surgical resection specimens of 27 cases, which, in 24 cases of differentiated adenocarcinoma, undifferentiated adenocarcinoma, poorly differentiated adenocarcinoma, 3 cases. Clinical stage Ⅰ ~ Ⅳ, according to the 1997 International Union Against Cancer (UICC) TNM staging criteria which stage Ⅰ + Ⅱ 13 cases, Ⅲ + Ⅳ period of 14 cases. 10 cases of extrahepatic bile duct dysplasia, are taken from the edge of the cancerous tissue. 5 cases of normal the biliary epithelial tissue as control. All specimens in 10% formalin fixed, paraffin-embedded, 4μm serial sections 10. Specimens HE staining, histological diagnosis by at least two pathologists. Dysplasia histologic diagnosis is based on the following criteria: cells arranged in disorder, loss of polarity, nuclear staining deep, uneven in size, nucleus and plasma imbalance, mild to moderate nuclear atypia, mitotic figures occasionally.
Survivin mouse anti-human polyclonal antibody was purchased from Fuzhou, a new biotechnology company. Smac rabbit anti-human polyclonal antibody purchased from Abcam companies. Albumen peroxidase labeled streptavidin (SP) staining kit, concentrated dimethyl benzidine (DAB) reagents, are Fuzhou Maixin biotechnology company.
SP method. The specific steps according to kit instructions. Survivin and Smac anti-working solution was diluted to 1:80 and 1:200. HCC tissues with the the Survivin protein known positive and Smac positive cardiac tissue as a corresponding positive control, phosphate buffered saline (PBS) instead of primary antibody as negative control.
1.4 immunohistochemistry the positive staining indicators judgment
Evaluation standard the Survivin positive staining judgment according to the literature [2,3]. Smac the positive staining judgment reference  Evaluation Standards. Each section observed from the site of the most positive staining, 400 times the light microscope, continuous observation of the five fields, divided into four grades according to the percentage of Survivin and Smac-positive cells: <5% for the negative (-); 6 50%为强阳性(+++)。">% to 25% of a weak positive (+); 26% to 50% for the medium positive (+ +);> 50% as strongly positive (+ + +).
1.5 statistical methods
SPSS10.0 statistical software for statistical The positive rate of the exact test and the chi-square test was used to compare. The correlation test using Spearman rank correlation analysis. P <0.05 was considered statistically significant.
2.1 extrahepatic bile duct dysplasia and carcinoma tissue expression characteristics
Survivin extrahepatic bile duct dysplasia and cancer tissue expression of positive expression mainly as a the cytoplasm pale yellow or yellow flakes or granular coloring, as shown in Figure 1.
Smac in normal bile duct epithelial tissue dysplasia epithelial tissue and cancer tissue were positive expression of cytoplasmic yellow or brown flake or granular coloring, as shown in Figure 2.
2.2 differential expression in dysplasia and carcinoma in the extrahepatic bile duct
Survivin expression in the three groups the difference was statistically significant (P = 0.049), Survivin expression in normal bile duct epithelial tissue and cancer tissue difference was statistically significant (P = 0.037), the Survivin expression differences in normal of biliary epithelial tissue and dysplasia not statistically significant (P = 0.580), dysplasia and carcinoma tissue Survivin expression difference was not statistically significant (P = 0.240).
Smac expression in the three groups, the difference was statistically significant (P = 0.028). Dysplasia and cancer tissue expression difference was statistically significant (P = 0.041), the difference was not statistically significant (P = 1.000), normal epithelial tissue and cancer tissue expression differences expressed in normal epithelial tissue and dysplastic tissue nor statistically significant (P = 0.057), as shown in Table 1. Table 1 normal biliary epithelial dysplasia and cholangiocarcinoma Survivin, Smac expression
2.3 Survivin and Smac dysplasia and carcinoma of the extrahepatic bile duct expression analysis
Dysplasia in 10 cases, one cases of survivin and Smac positive expression of positive expression located in almost the same tissues of patients with negative expression. Rank correlation analysis showed that Survivin and Smac in the extrahepatic bile duct dysplasia tissue expression was not correlated (r = 0.221, P = 0.540). 18 cases in 27 cases of cholangiocarcinoma survivin and Smac positive expression, four were positive in almost the same tissues. Rank correlation analysis showed that survivin and Smac expression in extrahepatic cholangiocarcinoma no correlation (r = 0.167, P = 0.406).
2.4 extrahepatic cholangiocarcinoma survivin and Smac expression with histopathological features and clinical stage comparison
Survivin and Smac extrahepatic bile duct, expression was no statistically significant difference in expression in differentiated carcinoma and poorly differentiated carcinoma, infiltration of blood vessels and nerves to extrahepatic cholangiocarcinoma lymph node metastasis in the expression of vascular nerve infiltration expression in the cancer tissue without lymph node metastasis was no significant difference I + Phase II and III + IV of the differential expression nor significant, as shown in Table 2. Table 2 extrahepatic cholangiocarcinoma Survivin and Smac expression and clinicopathological features Relations
Suzuki M, et al  of the extrahepatic bile duct reconstruction surgery or autopsy specimens of 12 patients with extrahepatic cholangiocarcinoma patients using three-dimensional computer reconstruction method analysis found that 75% of cases in the presence of dysplasia with cancer around confirmed the occurrence of the extrahepatic bile duct cancer with colon cancer and other malignancies as the development of cancer from dysplasia to carcinoma sequence. In this study, 27 patients with extrahepatic cholangiocarcinoma associated with 10 cases of dysplasia, accounting for 37.1% support the extrahepatic bile duct cancer occurred after precancerous lesions (dysplasia) to cancer of the pathological process.
Survivin belongs to the inhibitor of apoptosis protein (IAP) family, almost no expression in normal tissues and high expression in most tumor. The study of Survivin in the extrahepatic bile duct tissue next to the cancer, dysplasia and normal bile duct epithelial tissue expression of extrahepatic bile duct cancer tissue and adjacent normal bile duct epithelial tissue expression difference was statistically significant. Consistent with the findings of Evergreen . Survivin is highly expressed in the extrahepatic bile duct cancer can be partially involved in the development of extrahepatic cholangiocarcinoma Survivin by adjusting the extrahepatic bile duct cell apoptosis.
Lo Muzio L, et al  studied the normal oral mucosal dysplasia, abnormal proliferation of non-cancerous oral lesions, the development of cancer of the oral lesions and cancer organizations Survivin expression was 0,33%, 94% and 100%, respectively. Kawasaki et al  The immunohistochemical analysis Survivin in colorectal sequence and the expression, found with dysplasia cells to cancer cells transformed Survivin gene expression gradually elevated. In this study, using SP method detected adjacent normal epithelial tissue in the extrahepatic bile duct cancer, dysplasia and cancer tissue Survivin expression rates were 20%, 50%, 74.1%, an upward trend in the occurrence of extrahepatic cholangiocarcinoma in some carcinogenic factors stimulate the Survivin activation in normal tissue, so that the inhibitor of apoptosis, together with other carcinogenic factors, dysplasia, thus malignant transformation.
Survivin specifically high expression in the extrahepatic bile duct dysplasia stages and cancer tissue, can be partially represents Survivin expression increased cell biology into the intermediate links, to the malignant transformation of the cell biology of normal cells after dysplasia represents behavioral characteristics, the positive expression prompted organizations to learn still normal tissue there might dysplasia, representative of the excessive expression of Survivin cell malignant transformation possible. , Survivin expression may be elevated extrahepatic bile duct cancer early event, it’s too high expression represents cell malignant transformation may occur.
As the Survivin, X IAP IAP antagonist Smac gene has a role in promoting apoptosis. Smac ubiquitously expressed in normal human tissues, especially the highest, heart, liver, kidney, spleen, prostate and ovarian expression followed by brain, lung, thymus and peripheral blood lymphocytes express low expression in the adult testis tissue. Smac expression in tumor tissue is not much data, the expression of Smac neck cancer to raise , the Smac the downregulation of renal cell carcinoma . Yoo et al  Description and Smac expression level changes determined by tumor type, may be related to the heterogeneity of the tumor. The results of this study Smac in normal bile duct epithelial tissue has a higher expression (40%), slightly higher (50%) in the dysplastic tissue expression in cancer tissue was significantly higher (85.2%), in normal tissues and cancer expression differences, although not statistically significant, P = 0.057, close to 0.05, from a certain extent, it can be said Smac expression in extrahepatic cholangiocarcinoma expression in normal extrahepatic bile duct epithelium may be differences in the organization. Dysplasia and cancer tissue expression difference was statistically significant (P = 0.041 <0.05). Smac involved in the development of extrahepatic cholangiocarcinoma dysplasia to malignant transformation of cancer stage.
Smac expression in extrahepatic cholangiocarcinoma raised three kinds speculate: (1) based on Smac and Survivin interactions, mechanisms and the results of this study Survivin extrahepatic bile duct normal epithelial tissues, dysplasia and cancer tissue positive expression rate was gradually increased trend, suggesting the occurrence of extrahepatic cholangiocarcinoma development process, especially in cell malignant transformation stage, with the increased expression of Survivin body through some sort of signal transduction positive feedback caused Smac mRNA transcription enhancer, translation produce more Smac protein antagonizing Survivin antiapoptotic effect . The results of this study show that Survivin and Smac extrahepatic bile duct dysplasia and cancer tissues express no obvious correlation may be related to the small sample size. (2) detected in this experiment could be Smac mutants. Antagonizing Survivin mutants lost, lost the role to promote apoptosis, thereby promoting tumor development. (3) Smac there may be other roles outside in addition to promoting apoptosis. In short, Smac involved in the occurrence and development of the extrahepatic bile duct, and its mechanism of action pending further study.
This study shows that the survivin and Smac expression in extrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma degree of differentiation, and the presence or absence of blood vessels and nerves infiltration, lymph node metastasis and TNM stage. Santini D [12 to to to Survivin high expression (83%) found in ampullary cancer has nothing to do with the survival prognosis. Ikeguchi et al  reported Survivin expression can predict the prognosis of liver cancer patients after. The results of this study are inconsistent with the literature, may be associated with this experiment, the sample size, the type of tumor, but genetic testing as a new prediction method of tumor development and prognosis, and more and more to be confirmed. Survivin and Smac predictive significance of the biological behavior and clinical prognosis of extrahepatic cholangiocarcinoma, more experiments are needed to further study.