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Role of Cholecystokinin in the Perineural Invasiveness of Cholangiocarcinoma and Its Molecular Mechanism

Author: ZhengXiuHai
Tutor: WangShuGuang
School: Third Military Medical University
Course: Surgery
Keywords: cholangiocarcinoma perineural invasion cholecystokinin
CLC: R735.8
Type: PhD thesis
Year: 2005
Downloads: 88
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The incidence of the cholangiocarcinoma is rising in recent years. Up to now, surgery is still the main treatment. However, the resection rate of these patients with cholangiocarcinoma is very low because there is a special anatomy in the porta hepatis. The short-term tumor recurrence rate is also very high in most of the patients who undergo tumor resection. Besides the special anatomy of the bile duct, there are other reasons for this difficulty in the treatment of the cholangiocarcinoma and poor clinical effects. It includes the biological character of the cholangiocarcinoma. Metastasis of the cholangiocarcinoma appears easily in the early stage via many pathways. The tumor always invades the adjacent important organs when the symptoms appear and the patient comes to hospital. Perineural invasiveness (PNI) is one significant pathological character of the cholangiocarcinoma. It’s an independent pathway and different from the common metastasis pathway via lymph and blood vessels. The tumor cell can go up or down through the perineural interspace around the bile duct, and its incidence has an inverse correlation with the survival rate of the patients who undergo surgical resection of cholangiocarcinoma. There are aboundant peptidergic fibers around the bile duct, include the cholecystokinin (CCK) peptidergic fibers. Many gastrointestinal peptide hormones have important roles in the development of several digestive tract tumors by the autocrine, paracrine and neroendocrine. The CCK receptors are found in most cholangiocarcinoma cases, the PNI index of cholangiocarcinoma with positive CCK receptor is higher than that of cholangiocarcinoma with negative CCK receptor, and there are a lager invasive area and poor prognosis in the postive cases. It is found that CCK not only improves the growth of the cholangiocarcinoma, but also inhibits the apoptosis of the cholangiocarcinoma by up-regulating apoptosis inhibitor bcl-2 gene expression. Respecting the special anatomy of the bile duct and the close relationship with its surrounding nerves, it is important to study the function of the neuropeptides including CCK in the invasiveness and metastasis of the cholangiocarcinoma, especially in the perineural invasiveness. In this study, we investigated the function of CCK in the biological behaviour of the cholangiocarcinoma cell line QBC939 and its molecular mechanism, the function of CCK in the perineural invasiveness of the cholangiocarcinoma was also investigated. 1. Morphological observation of cultured cholangiocarcinoma cell line QBC939, the implantation metastasis characteristics of the detached cells and the function of CCK in this process. After the passage of the QBC939 cells, adherence, growth, splitting could be found by light microscope, and the circle or ellipse nest-like structures were formed. Following, the detatched cells in the culture medium gradually increased. With the scanning electron microscope, the villus were found decreased and became thick on the adjacent sides after the cells contacted, accompany with the decreasing contact area with other adjacent cells and the cultivate surface, Middle cells in the nest-like structures became round gradually and became detached cells at last. The apoptosis body was rare. In the CCK8s group, the culture cell density and the round cells with intensive stain were more than the control group. This phenomenon indicated that CCK8s improved growth of QBC939. With the scanning electron microscope, we found that most of the cell colony were irregularity, the cell surface was more smooth , the villus were shorter and thicker and with less villus between the cells in the CCK8s group than the cultured cells in the control group. The influence of CCK on the cholangiocarcinoma cell prompted that it may play an important role in the cell adherence and metastasis. Observation on the QBC939 conservation in suspending, cell counting and inoculation indicated that the detached cells were not anoikis or death, it could be keep alive in suspending for a long time, after re-attached on the suitable surface, it will return to original shape and continue to split and growth. Those result prompted that the re-attachment step of the detached tumor cells may be more difficult in clinical metastasis.There may be a same mechanism between the detaching–float-implantation-growth phenomenon of the cultured tumor cells in vitro and detaching-implantation-metastasis phenomenon of the cholangiocarcinoma in vivo. 2.The effects of CCK on cell adhesion, migration and invasion of cholangiocarcinoma cell line QBC939 were investigated. In this study, CCK8s was found to reduce the ability ofadhesion, increased the abilities of migration and invasion of QBC939 by dose-depending manner, and the physiological dose had a significant effect. The CCK receptor antagonist L18 and L60 resisted the role of CCK8s, and L60 showed stronger role than L18 on the assays of migration and invasion. These data indicated that CCK1 and CCK2 receptor have important role on the cell adhesion, migration and invasion of cholangiocarcinoma cell line QBC939, and CCK2 may play a stronger role than CCK1 in the migration and invasion of QBC939. 3.The mechanism of the bile duct cell invasiveness ability effected by CCK was investigated. In this study, we found that the expression of E-cad mRNA of QBC939 could be degrade by CCK8s, the Ct difference was 1.72 (3.29 times). And the expression of MMP-2、MMP-9 mRNA of QBC939 could be upgrade by CCK8s, their CT difference were MMP-2, 2.04(4.11 times),MMP-9, 3.55(11.71 times). The result of Western Blot show that the expression of E-cad protein could be degrade by CCK8s and the protein of MMP-2, MMP-9 could be upgrade. These finding indicate that CCK8s could promote insiveness and metastasis of cholangiocarcinoma by decrease the adhesion ability and increase the matrix degradation ability. Masson stain also show that there was an evident matrix degradation circle around tumor cells. By this method, the degradation of the matrix melted by tumor cell could be observed directly. 4. The effect of CCK on the perineural invasion of cholangiocarcinoma was investigated. In this study, the cholangiocarcinoma cell line QBC939 was implanted on the surface of the matrigel in which the mouse DRG was cultured. QBC939 cells were found to degrade and go through the Matrigel around the neurite, attach the neurite and grow around them. Cell masses circling the neurite formed. These finding indicated that The cholangiocarcinoma cell line QBC939 is neurotropism. The invasiveness of QBC939 is an active, continous process. After the CCK8s was added to the co-culture system of DRG/nerve cell and carcinoma cell, it could increase the invasiveness of QBC939. The CCK receptor antagonist L18 and L60 could resist the role of CCK8s, but there was not significance difference between L18 and L60. These results prompt that CCK could promote the nerve invesiveness of tumor cell, and this effect could be resisted by their antagonist. In this study, matrigel was found to satisfy the DRG/nerve culture. The process of the tumor cell going through the extracellular matrix and encroaching the neurite could be observeddirectly in this ststem. The DRG/carcinoma cell culture system was a suitable model to study the interreaction between nerve fibers and tumor cells. In conclusion, we found that the cholangiocarcinoma cell were anoikis resistance. The neurate invasiveness of the cholangiocarcinoma cell was an active, continous process. CCK had influence on the morphology of the cholangiocarcinoma cell line QBC939, and could promote its growth, reduce the ability of adhesion, increase the abilities of migration and invasiveness, and promote the Perineural invasiveness procedure. The mechanism was in relation to the CCK could degrade the expression of E-cad gene and upgrade the expression of MMP-2, MMP-9 gene.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Gallbladder, bile duct cancer
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