Dissertation > Excellent graduate degree dissertation topics show

Effect of Hepatitis C Virus NS5A Protein on TLR3,TLR4

Author: WenMing
Tutor: GongGuoZhong
School: Central South University
Course: Infectious Diseases
Keywords: HCV NS5A TLR3 TLR4
CLC: R512.63
Type: Master's thesis
Year: 2011
Downloads: 40
Quote: 0
Read: Download Dissertation

Abstract

The purpose of the hepatitis C virus (Hepatitis C virus, HCV) infection was the development of the global epidemic, the end is not one of the main reasons of the liver disease in Europe and the United States and Japan and other countries. At present, the global HCV infection rate of about 3%, about 170 million people infected with HCV, reached 35,000 cases of new-onset cases of hepatitis C each year. Of HCV infection rate of 1.5 to 3.5%, more than 30 million of the total infected people. Approximately 80% of HCV infection become chronic persistent state the liver chronic inflammation, necrosis and fibrosis, chronic infection can lead to development of cirrhosis and hepatocellular carcinoma, the patient's health and life hazards is very large, has become a serious social and public health problem. Non-structural proteins of the hepatitis C virus NS5A as an important non-structural proteins, attracting much attention in recent years for its research, it has anti-apoptotic transcriptional activation, interference with intracellular signal transduction pathways mediate levels of viral replication biological activity, suggesting that its forecast interferon therapy, viral replication, apoptosis, hepatocellular carcinoma and other aspects have an important role. TLRs interact with the body's mechanisms become one of the hot spots, including Toll-like receptor 3 (TLR3) expression in dendritic cells, dsRNA able to identify the virus plays an important role in the course of anti-viral. TLR4 is the earliest discovered the mammalian TLRs proteins, a variety of body cell surface can express TLR4, it is related to the gram-negative bacteria and endotoxin recognition and activation. Occurred in liver disease, including alcoholic liver disease, viral hepatitis, liver fibrosis, play an important role in the development process. In the present study, we transfected HCV NS5A protein expression plasmid to observe the impact of the HCV NS5A protein of liver cells TLR3 and TLR4 mRNA and protein expression the accumulated experimental basis for the pathogenesis of hepatitis C, and hepatitis C treatment provide a theoretical basis. Method 1. Plasmid transfection: QSG7701 cells were grown in six-well plates to be 60-70% cell fusion, transfection procedures in accordance with the instructions, respectively pcNS5A pRc / CMV plasmid transfected into cells, 12 hours after the change in DMEM medium containing 10% fetal bovine serum 48 hours after transfection, cells were collected for the next experiment. Immunocytochemistry: Immunocytochemistry for detection of TLR4 protein and NS5A protein expression. 3. RT-PCR: according to RNA extraction kit instructions, extracting the transfection of 7701 total cellular RNA, RNA concentration was measured with a UV spectrophotometer. Take 2ugRNA, reverse transcribed into cDNA was amplified by PCR, and then 2% agarose gel electrophoresis observations TLR3 mRNA and TLR4 mRNA and photographed. 4. Indirect immunofluorescence: TLR3 and TLR4 protein expression detected by indirect immunofluorescence method. 5. Western blot: β-actin as a reference to detect TLR4 protein expression using Western blot method. 1. The transfected plasmid group of pcNS5A 7701 cells within the expression of HCV NS5A protein particles, brownish yellow granules, mainly distributed in the cytoplasm, the nucleus around most obvious, untransfected and transfected with pRc / CMV plasmid group 7701 cells no HCV NS5A protein expression. Immunocytochemistry detection TLR4 protein expression results display transfection pcNS5A plasmid groups within TLR4 protein expression showed brown particles distributed in the cell membrane. Untransfected transfected and transfected with pRc / CMV plasmid group no significant TLR4 protein expression. Description HCV NS5A can promote the expression of the protein of TLR4. 3. TLR3 and TLR4 mRNA expression was detected by RT-PCR. The results show that, in reference to the expression of GAPDH within unanimous cases, pcNS5A plasmid group TLR4 mRNA expression was significantly higher than that of the empty vector group and untransfected group, empty vector group, and untransfected mRNA expression similar, the results suggest that HCV NS5A is able to activate the transcription level of TLR4 mRNA; However, pcNS5A plasmid transfection group TLR3 mRNA expression was no significant difference with the empty vector group and untransfected group, upregulates the HCV NS5A TLR3mRNA the transcriptional level at least. 4. TLR3 and TLR4 protein expression detected by immunofluorescence. Display the transfection pcNS5A plasmid group TLR4 protein expression showed green fluorescent particles, untransfected and transfected with pRc / CMV plasmid group had no significant TLR4 protein expression. Description HCV NS5A can promote the expression of the protein of TLR4. PcNS5A plasmid group transfection and untransfected groups and the transfected cells pRc / CMV plasmid group had no TLR3 expression of protein particles, indicating that HCV NS5A expression of TLR3 protein Upregulation. 5. TLR4 protein expression detected by Western blot, β-actin as an internal reference. The results showed that pcNS5A plasmid transfection group TLR4 protein expression was significantly higher than that of the empty vector group and untransfected group, similar to the expression of the empty vector and untransfected. Description HCV NS5A can promote the expression of the protein of TLR4. Conclusion 1.HCV NS5A expression plasmid transfection QSG7701 cells expressing HCV NS5A protein. 2. HCV NS5A can activate TLR4 mRNA transcription level. 3. HCV NS5A can promote TLR4 protein expression. 4. HCV NS5A expression of the TLR3 mRNA transcription level and TLR3 protein had no significant upregulate.

Related Dissertations

CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis C (non- A non-B )
© 2012 www.DissertationTopic.Net Mobile