| Objective: To investigate the expressions of Erb B4 in human intrahepatic cholangiocarcinoma(ICC) tissues, adjacent tissues and intrahepatic bile duct tissue.Analysis of the relationship between clinical significance and the expression of Erb B4 in cholangiocarcinoma. Further to observe the effect of Erb B4 inhibitor on proliferation and apoptosis of cholangiocarcinoma cell line QBC939. Research the mechanism of Erb B4 in advancing the progression and metastasis of the cholangiocarcinoma preliminary, and provide ideas for clinical treatment and prognosis of intrahepatic cholangiocarcinoma.Methods: 1.The tissue samples and clinical data were collected from patients in Human People`s Hospital from April 2010 to November 2014,which were undergone surgery. Immunohistochemical methods were used to detect the expressions of Erb B4 in bile duct tissues in three groups, tumor group(carcinoma tissues from 30 cases of ICC, have pathological detection for intrahepatic cholangiocarcinoma), adjacent group( Distance carcinoma outside the 1cm organization from the same 30 cases of ICC, have pathology detection for non cancer tissues) and calculus group(bile duct tissues from 10 cases of intrahepatic bile duct stones, have pathology detection for non cancer tissues). SPSS 20.0 was used to analyze the differences among the three groups and the relationship between the expression of Erb B4 and various clinicopathological parameters.2. Human cholangiocarcinoma cell line QBC939 was cultured in vitro. Detect the expression of Erb B4 in QBC939 cells by Immunocytochemical methods. Evaluate with MTT assay the effect of Tyrphostin AG1478 of different times(24h,48 h,72h) and different concentrations(0, 0.1μg/ml, 1μg/ml,10 μg/ml,100 μg/ml) on the growth of QBC939 cells. Measure the cell cycle and apoptosis by flow cytometry(FCM) after the 72-hour treatment of Tyrphostin AG1478(20 μg/ml).Results: 1. ICC group showed significantly higher over expression rate of Erb B4 compared with adjacent group( 83.3% vs 36.7%, p <0.05) and calculus group( 83.3% vs 20.0%, p <0.05). And the differential expression of Erb B4 mainly located in the nucleus.The expression of Erb B4 in the nucleus, ICC group was significantly higher than the others( p <0.05); but in the cell membrane and cytoplasmic,there was no significant difference between the three groups. 2.The over expression of Erb B4 in ICC was correlated with lymph node metastasis and TNM staging( both p <0.05). 3.Immunohistochemitry(IHC) indicates that Erb B4 shows positive expression in QBC939 cells, mainly in cellular membrane,cytoplasm and nucleus is also a small amount of expression. The expression of Erb B4 had no noticeable change after treatment by inhibitor Tyrphostin AG1478. 4. Tyrphostin AG1478 can inhibit the proliferation of QBC939 cells,and the higher the concentration of inhibitor and the longer the intervention time, the inhibition was more significant.The IC50(24h) is 55.98 μg/ml, IC50(48h) is 32.48 μg/ml, IC50(72h) is 20.85 μg/ml. 5. Flow cytometry showed that intervention of Tyrphostin AG1478(20 μg/ml) after different times, QBC939 cells cycle is arrested in S phase.The percentage of S phase in the control group is 11.264%, group 24 h is 14.764%, group 48 h is 19.934%, group 72 h is 39.392%. The apoptosis rate of QBC939 cells increased gradually, the apoptosis rate inthe control group is 4.503%, the 24 h group is 9.145%, the 48 h group is 14.219%, the 72 h group is 17.499%, there are statistically significant compared with the control group.Conclusions: 1. Erb B4 protein is expressed in the cell membrane, cytoplasm and nucleus in intrahepatic bile duct tissues,and in ICC was mainly expressed in the nucleus. 2.The over expression of Erb B4 in ICC were related to lymph node metastasis, and TNM staging. 3. Tyrphostin AG1478 can inhibit the QBC939 cell proliferation which expression of endogenous Erb B4,and induce cell cycle arrest in S phase, and induce its apoptosis; but could not inhibit the expression of Erb B4 in QBC939 cells. |
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