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Stable Transfection of Hepatitis B Virus X Gene into Cholangiocarcinoma Cell Line RBE and Its Effects on Biological Behavior

Author: LvTao
Tutor: LiuHouBao
School: Fudan University
Course: Surgery
Keywords: Hepatitis B Virus X protein cholangiocarcinoma lentivirus vector cell proliferation
CLC: R512.62
Type: Master's thesis
Year: 2011
Downloads: 50
Quote: 0
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Abstract


Hepatitis B Virus (HBV) belongs to the prototype of hepadnavirus family. HBV infection leads to acute or outbreak hepatitis, chronic hepatitis, and is closely associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). It is estimated that 350 million people are carriers of HBsAg all over the world, among which one third are Chinese, there are 750 thousand people died from disease infected with HBV, among which 280 thousand come from Chinese. HBV infection has become an extremely severe problem of public health.HBV X gene and its expression product may play a crucial role in the development of HCC related to HBV infection. Although lacking the ability to bind to DNA, HBx protein can interact with a variety of host proteins and affect their functions which will bring about a great diversity of biological effects. In order to maintain cellular homeostasis, cells will start independent ordered dead controlled by genes, this process is called apoptosis which involved in a series of genetic activation, expression and regulation. Current studies discover that some kinds of tumors are not induced by unlimited proliferation, and that apoptotic pathway blocking or apoptotic ability losing has attributed significantly to tumorigenesis. Foreign and domestic previous study revealed that, in some experimental system, HBx protein was able to inhibit apoptosis by suppressing p53 and Fas signal passway or inactivating caspase-3. However, at the same time, other experiments showed that HBx was able to induce apoptosis, the exact mechanism is still unclear at present.Recent study reported that HBV infection may relate to the development of cholangiocarcinoma and may be one of the risk factors of cholangiocarcinoma. For example, although exact evidence that HBV was able to infect bile duct epithelial cells or to complete viral life cycle has not acquired yet, HBx protein has been detected in some cholangiocarcinoma tissue slice, and its positive rate was higher than HBsAg. Some investigations showed that after transient transfection, HBV X gene enhanced proliferation of cholangiocarcinoma cells. Nevertheless, other experiments observed that HBV X gene promote apoptosis after transfection.In order to study the possible relationship between HBV and the development of cholangiocarcinoma, our study constructs recombinant lentivirus expressing HBsAg, HBc/eAg and HBx protein respectively, and we also realized high-efficient expression after recombinant lentivirus infecting RBE cholangiocarcinoma cells. Based on the infected RBE sells, we further established RBE cell lines stable expressing HBsAg and HBc/eAg by utilizing fluorescence activated cell sorting.Our study also preliminarily analysis the influence of biological property of RBE cells by expressed HBx mediated by recombinant lentivirus. The results of cellular growth curve and clone formation test revealed that HBx exerts infaust effect on the proliferation of RBE cells, however, apoptosis analysis has not detected significant proapoptotic or antiapoptotic effect yet.Though the above-mentioned works, on the one hand, we provide a set of high-efficient methods and tools to investigate the effect of HBV protein on bile duct epithelial cells. On the other hand, we provide clues and thread to further study the possible actions of HBxAg on the development of bile duct tumor through revealing the influence of HBxAg on biological property of RBE cells.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B
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