Significance And Expression Of Microvessel Density, VEGF, VEGF Receptor-2 And Nuclear Factor-kappa B Cholangiocarcinoma

Bile duct carcinoma is one of the most malignant cancers, the 5-year survival is only 10%. Operation is the only effective way. Although the situation has been improved with the progress of diagnostic radiology and surgical techniques, the curative effect can not been improved greatly because the special anatomatic position and the invasion characteristic of bile duct carcinoma. So we have to go deep into the mechanism and find new methods.Angiogenesis, many studies have revealed that the growth of new capillary blood vessels, is a key step in tumor growth and metastasis. It is growth out of control and imbalance of enhancing factors and inhibitors. Vascular endothelial growth factor (VEGF) is a most popular enhancing factor; it combines with its specific receptor and produces biological effects by receptor phosphorylation. Studies have revealed that VEGF increased in breast cancer, prostate cancer and the level was correlated with the clinical stage and prognosis. Nuclear factor-kappa B (NF-kB) is one of the most important transcription factors. Researches over the last few years have revealed that NF-kB affected tumor progression. It could regulate correlative genes of angiogenesis and also affect apoptosis. There have been few studies on angiogenesis in bile duct carcinoma by now.In our study, we determined microvessel density (MVD), VEGF and its receptor (Flk-1/KDR), and NF-kB of bile duct carcinoma in 50 cases throughimmunohistochemistry. And associated with pathological characteristics and follow-up data, we discussed the effect of angiogenesis in bile duct carcinoma and its possible mechanism. It would offer a method to value the malignancy and prognosis of bile duct carcinoma, and also a base for further study.Material and MethodsPatients: Paraffin embedded specimens from 50 patients who underwent operation for bile duct carcinoma in the Sir Run Run Shaw hospital or the Second affiliated hospital of Zhejiang University medical college from 1997 to 2003 were selected for this experiment. In all selected patients, 27 cases received radical operation and the others received palliative operation. There were 27 male and 23 female. The mean age was 59.3 years (32-84). All specimens were adenocarcinoma, and well-differentiated adenocarcinoma 19 cases , moderated-differentiated 21 cases, poor-differentiated 10 cases; with lymph nodes metastasis 13 cases; with nerve invasion 22 cases; with pancreatic invasion 14 case. The control cases were from liver donors for transplantation.Experiment grouping: the normal tissues were from 9 cases of liver donors for transplantation; the para-tumor tissues were from the incising margin of radical operative specimens and tumor tissues were from 50 cases operative specimens.Follow-up: 27 cases who received radical operation were followed up by phone, letters and out-patient clinic way. We found out the survival time and the death reason.We determined the MVD, VEGF165, F1k-1/KDR and NF-kBp65 expression by immunohisto- chemical En Vision method. Judging and grading system: 1 MVD measure: vascular endothelial cell was immunostained yellow brown by antibody of CD34 and every positive immunostained endothelium was considered as one microvessel. Under high power lens (10*40), counting microvessels in 5 hotspots and taking the mean as MVD. 2 The expression of VEGF165, Flk-1/KDR and NF-kBp65: foreach antibody, positive immunostaining appeared as yellow brown in cytoplasms. According to Frank's semi-quantity method, selecting 5 high power fields, observing 100 cells in every field, and counting scores of the immunostained intensity and the percent of positive cells. The standard as followed: (1) the percent of positive cell <5% 0 score, 5%~25% 1 score, 26%~50% 2 score, 51%~75% 3 score, >75% 4 score. (2) the staining intensity: no stained 0 score, mild stained 1 score, moderate stained 2 score, deep stained 3 score. Adding (1) and (2), 0 score is negative, 1~3 score is mild positive(+), 4~7 score is strong positive(++).Statistical analysis: All data was p...