The Expression Of BDNF And TrkB In Cholangiocarcinoma And Effects Of BDNF On Cholangiocarcinoma Cell Line QBC939

Chapter 1 Expression and clinical significance of BDNF and TrkB in CholangiocarcinomaObjective:To explore the expression of BDNF and TrkB protein in normal bile duct and cholangiocarcinoma.To analyze the relationship between BDNF and TrkB expression and the clinical pathological features in cholangiocarcinoma.Methods:The expression of BDNF and TrkB was detected by immunohistochemistry in 10 cases of normal bile duct and 42 cases of cholangiocarcinoma.Results:Normal bile duct were all negative for BDNF and TrkB protein expression,however,in cholangiocarcinoma,the positive rate were 71.4%(30/42)and 69.0%(29/42),respectively.BDNF positive rate were 92.9%,90.0%,93.3%in poorly differentiated group,node lymph metastasis group and distant metastasis group,whereas 60.7%, 54.5%,59.3%in well and moderately differentiated group,no lymph node metastasis group and no distant metastasis group.TrkB positive rate were 100%,90.0%,93.3%,in poorly differentiated group,lymph node metastasis group and distant metastasis group,whereas 53.6%,50.0%,55.6%in well and moderately differentiated group,no lymph node metastasis group and no distant metastasis group.BDNF and TrkB positive rate had no significant relation with age,gender and the size of tumor.There were positive correlation between BDNF and TrkB(r=0.736,p＜0.01).Conclusion:BDNF and TrkB protein were over appeared expression in cholangiocarcinoma,but were not appeared in normal bile duct.The positive expression of BDNF and TrkB had related with poorly differentiated,lymph node metastasis and distant metastasis,and there were positive correlation between BDNFand TrkB.Chapter 2 Effects of BDNF on Cholangiocarcinoma cell line QBC939Objective:To investigate the effects of exogenous BDNF on biological behavior and expression of FAK of Cholangiocarcinoma QBC939 cell line.Methods:control group,low concentration group,middle concentration group and high concentration group were cultured with RPMI 1640,and RPMI 1640 containing 25ng/ml,50ng/ml and 100ng/ml BDNF,respectively.(1)BDNF and TrkB were expression in QBC939 were analyzed by immuocytochemistry;(2)Cell proliferation was estimated by MTT assay;(3)Establish suspension culture model by culture plate treated with Poly-HEMA,the cell anoikis rate was analyzed by flow cytometry(FCM);(4)Adhesion ability was analyzed by adhesion assay;(5)Invasion ability was analyzed by Transwell chamber assay;(6) Expression level of mRNA and protein of FAK were investigated by RT-PCR and Western Blot.Results:(1)Immuocytochemistry staining showed:there were expression of BDNF and TrkB protein in QBC939 cell line.(2)MTT assay showed:BDNF can promote cell proliferation,50ng/ml,100ng/ml more than 25ng/ml,but there were no significantdifference between 50ng/ml and 100ng/ml;(3)Flow cytometry(FCM)showed:BDNF can enhance the ability to resist anoikis,but there were no significant difference among different group.(4)Adhesion assay showed:BDNF can enhance the ability of adhesion,but there were no significant difference among different group.(5)Transwell chamber assay showed:BDNF can enhance the ability of invasion in concentration-dependent manner. (6)RT-PCR and Westem Blot showed:BDNF can up-regulate the expression of mRNA and protein of FAK,respectively,but there were no significant difference among different group.Conclusion:1.BDNF and TrkB protein were expressed in cholangiocarcinoma QBC939 cell line;2.Exogenous BDNF can promote cell proliferation,as well as enhance the ability to resist anoikis and the ability of adhesion and invasion in cholangicarcinoma QBC939 cell line; 3.Exogenous BDNF can up-regulate the expression of FAK in QBC939 cell.Chapter3 Effects of BDNF on transplanted tumor of Cholangiocarcinoma cell line QBC 939 in nude miceObjective:To investigate the effects of exogenous BDNF on the growth of transplanted tumor of Cholangiocarcinoma QBC 939 in nude mice.To investigate the effects of exogenous BDNF on the expression level of FAK.Methods:Establish mouse transplanted tumour model.0.9%sodium chloride solution and BDNF were injected subcutaneously into nude mice of control group and BDNF group respectively.(1)To observe the growth rate.The size and weight of tumor were measured;(2)To observe the histological morphous by HE staining;(3)The expression of BDNF and TrkB were detected by immunohistochemistry;(4)To investigate expression level of mRNA and protein of FAK by RT-PCR and Western Blot.Results:Transplanted human cholangicarcinoma were developed in all nude mice.(1)Growth of tumor was significantly promoted by BDNF.The tumor volume and weight in BDNF group were more than control group significantly.(2)HE staining show typical histological morphous of,cholangicarcinoma of transplantation tumor tissue. (3)Immunohistochemical staining show:there were expression of BDNF and TrkB protein in transplantation tumor tissue.(4)RT-PCR and Western Blot show:BDNF can up-regulate the expression of mRNA and protein of FAK in transplantation tumor.Conelusion:1.Exogenous BDNF can promote the growth of transplantation tumor.2.There were expression of BDNF and TrkB in transplanted tumor tissue.3.Exogenous BDNF can up-regulate the expression of FAK in transplanted tumor.