| Chapter I. CXCR4Over-expressed in Extrahepatic Cholangiocarcinoma and Correlatated with Lymph Node Involvement and Neural Invasion.Background:Extrahepatic cholangiocarcinoma (extra-HCC) was identified as biliary carcinoma that originated from biliary tract epithelial of left or right hepatic biliary tract to common biliary duct. Extrahepatic cholangiocarcinoma locates most commonly at the proximal bile tract. extra-HCC is an aggressive carcinoma, with features of extra-hepatical invasion, neural invasion and lymph-node metastasis. The effect of adjuvant radio-chemotherapy on HCC was not ideal, and radical resection was the only cure. The prognosis of extra-HCC is poor. Lymph-node metastasis and neural invasion was reported as the most predictive factors for poor prognosis of extra-HCC. The effect of chemokine and receptor was reported to have played a critical role in the proliferation and migration progresses in many kinds of tumors. CXCR4is the specific receptor of chemokine SDF-1, which is widely detected in target migrating organ such as lung, bone, liver and peripheral tissue as well. Previous studies reported that CXCR4over-expression and the CXCR4-SDF-1axis was important promoting factors for carcinoma cells to migrate and to infringe spcifically. However, the relation of CXCR4over-expression and neural invasion and metastasis in HCC need to be further investigated thoroughly.Purpose:To investigate the CXCR4expression in extra-HCC, cholangitis, and normal biliary tract tissue; and analysis the association between the over-expression of CXCR4and clinicopathological factors in extra-HCC patients.Method:43of perihilar cholangiocarcinoma, cholangitis and normal biliary tract tissue specimens were collected from patients that hospitalized in XiangYa hospital in2009-2011. Immunohistochemical staining was applied to normal hilary biliary tissue, benign and malignant biliary tract specimens to evaluate CXCR4expression respectively; clinicopathological varieties of etxtra-HCC patients were documented. Paired/analysis was used to investigate CXCR4over-expression in different specimens in HCC, cholangitis and normal biliary tract tissue; chi-square analysis was used to study the relation between CXCR4over-expression and the clinicopathological features.Results:CXCR4was detected in65.22%cholangiocarcinoma and in28.57%cholangitis tissue specimens, CXCR4expression didn’t express in normal biliary tract tissue specimens (0/6); CXCR4over-expression was only detected in extra-HCC tissue. Statistical analysis demonstrated that CXCR4over-expression was related to neural invasion and lymph-node metastasis in HCC (p<0.05).Conclusion:CXCR4over-expression was only detected in HCC tissue; it was suggested that CXCR4over-expression was association with neural invasion and lymph-node metastasis.Chapter Ⅱ. Establishing of in Vitro Cholangiocarcinoma Neural Invasion Model by Co-culture Assay Using Induction of ESC DifferentiationBackground:In order to investigate the progression and mechanism of tumor migration, colonization and distant metastasis, the interactions of tumor cells and in situ microenvironment were studied thoroughly. However a useful and efficient model is urgently needed for investigating the behavior and mechanism of neurotrophic invasion of extrahepatic cholangiocarcinoma. Human embryonic stem cells were pluripotent stem cell; it can be induced into different mature differentiated cells by certain stimulation factors including neuron and glial cells. In vitro co-culturing using Transwell chamber was widely used for studying tumor invasion process. Extrahepatic cholangiocarcinoma cells co-cultured with neural cells in Transwell chamber was desgined to mimic and investigate neural invasion in cholangiocarcinoma.Purpose:To committed induce embryonic stem cells and obtain neural cells; and to establish a novel and efficient co-culture perineural invasion model for extrahepatic cholangiocarcinoma.Method:Human embryonic stem cells were induced by glial cell-derived neurotrophic factor and retinoic acid to form neural cells. Immunocytochemical staining was used to detect neural markers synaptophysin (SYN) and glial fibrillary acid protein (GFAP) of the induced cells. In the experimental group, cholangiocarcinoma cells were co-cultured with neural cells in the upper chamber of the Transwell plate, while in he control group cholangiocarcinoma cells were cultured alone. Neurotropic growth was observed in microscope. Paired student t analysis was adopted to compare the counted cholangiocarcinoma that penetrated through membrane.Results:Formation of neurospheres and neural-like cells were observed following induction at24and48hours, respectively; synapses were viewed to protrude from neural-like cell bodies after incubation for96hours.48hours after incubation, immunocytochemical staining of the cells showed that synaptophysin and glial fibrillary acidic protein were expressed in the neuron-like cells and gliocytes-like cells, respectively. Extrahepatic cholangiocarcinoma cells penetrated through matrigel/polyethylene membrane from the upper chamber to the lower chamber of the Transwell in the co-culture group more expediently than those in the control group (68±8.3/field versus46±5.7/field, P<0.05). Conclusion:The novel model is a valuable tool to study the neurotropic gowth of extra hepatic cholangiocarcinoma.Chapter III. Proliferation and Neurotropic Growth in extra-HCC Cells was Inhibited by CXCR4SilencingBackground:Small interfering RNA (siRNA) is a kind of short double chains RNA fragments. siRNA can specifically bind to and degradate target nucleotide fragment; via virus infection or artificial insertion; and block the transcription and translation process of target mRNA efficiently and specifically. RNA interferencing (RNAi) is becoming a novel effective method and tool for gene therapy, gene structure and functions researching. CXCR4was detected over-expressed in colorectal cancer, pancreatic carcinoma, salivary gland carcinoma and many other tumor cells. A series of proliferation, migration, survival and metastasis progression was deemed to have been initiated, promoted, or, adjusted by CXCR4function. SDF-1is the only ligand to CXCR4. SDF-1is widely expressed in various tissue cells, including perineural cells. The speculation of SDF-1-CXCR4axis has played a critical role in neurotropic growth and neural invasion in cholangiocarcinoma need to be verified and studied thoroughly.Purpose:To investigate the extrahepatic HCC cell proliferation and frequency of neurotropic growth influenced by RNAi mediated CXCR4silencing.Method:CXCR4-siRNA was transfected in to extra-HCC cell QBC939and SNU-1196by Lipofectamine2000; transfection ratio was observed and counted by Fluorescence microscopy. WesternBlot immunoblotting and real-time PCR was used to investigate the effects of CXCR4-siRNA mediated CXCR4silencing in cholangiocarcinoma cells. SDF-1expression on the h-ESCs derived neural cells membrance was detected by ELISA assay. Neural cells and cholangiocarcinoma cells were co-cultured in Transwell chamber; and the cholangiocarcinoma neural invasion ability was evaluated by comparing the number of penetrated HCC cells in silencing and control group. Cell proliferation was determined Methylthiazolyl tetrazolium (MTT) and flow cytometry (FCM) analysis.Results:CXCR4-siRNA was successfully transfected into extra-HCC cells; the transfection rate is80%. CXCR4and CXCR4-mRNA expression was suppressed in both HCC cell lines QBC939and SNU-1196(P<0.05). CXCR4-siRNA number370present the most overwhelming efficiency. Extra-HCC and neural cells co-culture migration assay depicted that neurotropic growth and invasiveness in cholangiocarcinoma cells was significantly depressed by CXCR4-siRNA interferencing (P<0.05evenly). Though there is no statistical significance, QBC939and SNU-1196cells in transfection group showed relatively reduced proliferation rate. The apoptosis rate was raised to54.63%and48.25%, in extra-HCC cells QBC939and SNU-1196, compared to control group. Conclusion:CXCR4involved in neural invasion and proliferation of HCC cell lines QBC939and SNU-1196, indicating that CXCR4could be promising therapeutic target for perihilar cholangiocarcinoma. |
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