Study On The Expression Of Angiostatin And CD34 In Cholangiocarcinoma And Clinical Significance

Study on the expression of angiostatin and CD34 in cholangiocarcinoma and clinical significanceObjectsInhibitors of angiogenesis, such as angiostatin, are increasingly used for targeting the tumor neovasculature and have had mixed success. Angiogenesis is a prerequisite for growth and differentiation of organs and tissues, i. e. sprouting of new vessels, their remodelling and regression. It is involved in many pathological processes, particularly growth and metastasis of tumors. Angiostatic therapy is a promising new strategy in the treatment of cancer. Angiogenesis inhibitors could intervene in the different phases of the angiogenic cascade, i. e. migration, proliferation, differentiation and three-dimensional organisation of endothelial cells, to inhibit the generation of tumor vessels. The target of antiangiogenic cancer treatment is the genetically normal endothelial cell, therefore, the development of resistance to angiostatic therapy is very unlikely, so inhibiting tumor angiogenesis is a promising new strategy for treating cancer. About tumor antiangiogenic inhibitors have been reported, of which endostatin, a newly found potent endogenous inhibitor of angiogenesis, has the best effect on tumor angiogenesis. Recently, great attention has been paid on the elation between angiogencsis and the pathogenesis, development of the tumor. Not only the new blood vessels bring nutrition to the tumors, but they also give the tumor cells to be transferred. A great deal of study shows that there is a high relationship between angiogenesis and tumors metastasis. The microressel density (MVD) of the tumor tissues not only reflect the growth of the tumor blood vessels quantitatively, but can also predict the progress, metastasis and recurring of the tumour. CD34 are often used to be the markers of MVD. In this study, we detected the expression of angiostatin by in situ hybridization and CD34 in cholangiocarcinoma tissue by immunohistochemisty method, and analyzing their correlation, then discussing their roles in the pathogenesis, development, invasion and metastasis in cholangiocarcinoma to provide the new ideas and theory basic for diagnosis, therapy and prevention.Methods1. Materials Forty-five cholangiocarcinoma patients for operation during 2004 and 2006 were selected, 18 of which were men and 27 were women. They aged from 18 to 76. All patients didn't have radiotherapy and chemotherapy before operation. All the resected samples were pathologically detected to determine differentiation, histological type, and tumor staging.2. Methods: Angiostatin was detected in situ hybridization method in 45 cases of cholangiocarcinoma tissue and 11 cases of adjacent normal tissue. AS in situ hybridization kit was used for detection. The sequence of AS oligonucleotide probes asfollows: 1: 5'--GCACT TGGCT GTTGG TTGTA TGGCA C; 2: 5'--CGTCA GGATT GCGGC AGTAG TTTTC AT; 3: 5'--CTGCT CTTTG CTGTG GTACT GGAAT GAC The expression of AS mRNA was assessed by Meta Morph Imaging System depended on gray values. Stained with orange in the cytoplasm was determined as positive (+). The intensity of staining were divided into five degrees: (-) no staining (gray value＞130); (±) weak staining (gray value 125-130); (+) moderate staining (gray value 115-125); (++) strong staining (gray value 105-115); (+++) very strong staining (gray value＜105).And immunohistochemistry was used in the MVD calculation by vascular endothelial cell specific antigen CD34. Immunohistochemistry: Samples were put into 3% H₂O₂, and antigens were repaired in microwave oven by 10 minutes in 95-96℃. All samples were blocked in 10% goat serum for 30 minutes. First and secondary antibody was put onto the slices consecutively, and then SABC and DAB were put on them for color display. Standard: (-) the ration of positive cells＜5%; (+) 5%-20%; (++)＞20%. (+) and up were considered as positive.The relationship of AS expression and MVD with the tissue invasion, lymphatic metastasis and prognosis of cholangiocarcinoma was analyzed, respectively.Results1. Expression of AS in different tissues: Of all the 45 patients, the positive of AS was 60.0% (27/45) in cancer tissues, and 18.2% (2/11) in normal tissues, respectively. The expression of them in cholangiocarcinoma tissues were more than that in normal tissues (X²=6.19, P＜0.05) (See Table 1.).2. There was a close relationship between the expression level of AS and whether there was lymph node metastasis (P＜0.05).3. MVD was significantly higher in the cancer group (31.78±4.41) than that in normal group (26.80±5.14, P＜0.05) (See Table 3.).4. MVD counting value in low differentiated cholangiocarcinoma was higher than that of high differentiated cholangiocarcinoma patients, P＜0.05. MVD counting value in patients with tissue infiltration of hilus hepatic (37.79±10.15) and lymph node metastasis (39.22±7.75) was higher than that in patients without tissue infiltration of inferior segment of bile commen duct (33.42±8.99) and no lymph node metastasis (32.62±7.96), P＜0.05.Conclusion1. AS is higher expression in cholangiocarcinoma tissue than normal group, and expression of AS is correlated with lymph node metastasis. AS plays an important role in development of cholangiocarcinoma.2. CD34 was highly expressed in cholangiocarcinoma tissues, and expression of CD34 is correlated with lymph node metastasis, differentiated degree and position of tumor.3. AS has close relationship with the angiogenesis of the cholangiocarcinoma, and may contribute to the restraint of tumor invasion as well as metastasis in cholangiocarcinoma. AS expression and MVD value are markers for the invasion, metastasis and prognosis of cholangiocarcinoma.