Dissertation > Excellent graduate degree dissertation topics show

Mechanism of Hepatic Steatosis Induced by HCV core Protein

Author: JiangLiNa
Tutor: LiQing；YeZuo
School: Fourth Military Medical University
Course: Pathology and Pathophysiology
Keywords: HCV core 3α HCV core 1b hepatocyte steatosis
CLC: R575.5
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 0
Read: Download Dissertation

Abstract

Hepatitis C virus (HCV) infection is one of the serious global publichealth problems. There are 17 million patients infected with HCV in the world.Of these patients, 80% develop into chronic hepatitis c. At least 30% of thembecome more severe liver diseases, such as fatty liver, cirrhosis and livercancer.HCV belongs to the yellow virus family, the virus particles own 9.6 kbpositive single-strand RNA. The open reading frame can form a single proteinbody including 3,000 amino acids. This protein can be adherent to the ER,The protein is transformed into three structure protein (core, E1, E2) and 7nonstructural protein (p7 NS3, NS4A, NS2, NS4B, NS5A, NS5B,) with thehelp of proteases of the host and the virus. HCV virus can be divided intoseven main genotypes and more than one type. The virus may bind to thespecial proteins in the surface of the cell, and then start the life cycle of thevirus through endocytosis. The HCV core protein is unglycosylated protein which is22kDa.It isencoded by an open reading frame which is close to 5`protion. As a typicalflariviral virus, HCV core protein contains amount of lysine and arginine,especially in the N terminal). It combined with RNA genome in order to formHCV virus nucleocapsid. HCV core protein`s sub-cellular localization is inthe cytoplasm, but when the C terminal is missing, it can transfer to thenucleus in cell. This suggests that HCV core protein`function in HCVpathogenic process may be direct.Long-term chronic HCV infection HCV virus can affect the body lipidmetabolism. It can lead to the accumulation of lipid droplets in the liver.Studies have shown that intracellular lipid droplets in the HCV viral life cyclealso play a very important role.HCV can cause fatty liver, liver cirrhosis and liver cancer eventually. Thepathogenesis has not been fully clarified. The prevailing view is that the fattychange is the disorder of host cell lipid metabolism. This process has playedan important role in liver fibrosis.HCV core protein is an important risk factor in steatosis. It involves theaccumulation of lipid droplets, changes in gene expression of fat andfat-related protein. Host gene`s expression help to promote virus life cycle.Abnormal liver lipid metabolism accelerates the liver diseases, whilelipid droplets play a key role in the process of HCV replicatio. Researchhcv-infected induced the molecular mechanism of fatty liver is particularlyimportant. Analysis the pathogenesis of fatty liver induced by HCV, controlthe HCV infection, thus inhibiting the formation of steatosis, at last thedisease is prevented. This is one of the hotspots of the world. HCV core protein 3a can strongly induced the liver steatosis. There areonly a few different base pairs between HCV core 3a and other genesequences. The difference may be the key point of the mechanism of steatosis.This study focuses on the mechanism of core protein of HCV subtype 3ainduced steatosis. We constructed a HA-HCV core 3a and HA-HCV core 1bfusion gene in adenovirus. They infected mouse primary hepatocytes. After24h, 48h, 72h, morphological characteristics were observed in hepatocytesinfected with HCV core 3a and HCV core 1b. We prepare to reversetranscription and real-time quantitative PCR in order to detect mRNAexpression of host cell receptors PPAR-α, host genes ACC1 and FAS whichassociated with the lipid metabolism. Further exploration of molecularmechanism of hepatic steatosis induced by the HCV core protein is needed.Objectives1. Morphological changes of lipid droplets in primary hepatocyteinfected by HCV core 3a protein adenovirus;2. Real-time PCR detect of PPAR-α, ACC1 and FAS.Methods1. Construction of HA-HCV core 3a and 1b adenovirus fusion gene;2. Isolation of mouse primary hepatocyte;3. Adenovirus infection of mouse primary hepatocytes;4. Exam of the primary hepatocytes by immunofluorescence detectionof hepatocyte in morphological characteristics; 5. Culture of liver cell line AML12, then the adenovirus infection;6. Real-time quantitative PCR detection of the mRNA of the cells.Results1. We successfully constructed pAdEasy-HA-HCV core 3a and 1badenovirus.First, HCV core 3a (1b) fragment were connected with pshuttle-CMVvector. Shuttle-plasmid was homologously recombinated with pAdEasy-1adenovirus vector. Recombinated plasmid was transfected with AD293 cells.After packaging, high-titer recombinant adenovirus was amplified. The viruswas successfully detected by western blot.2. Primary mouse hepatocyte was infected with pAdEasy-HA-HCV core3a adenovirus. The size of the lipid droplet is larger than the numberof that infected with 1b.The primary mouse hepatocytes were cultured. The infectionpAdEasy-HA-HCV core 3a and 1b adenovirus in mice hepatocytes inducedmorphological differences after 24h, 48h and 72h. We found that,pAdEasy-HA-HCV core 3a formed larger and more lipid dropletsthan 1b.Subtypes 1b can be induced little more lipid droplets than normal liver cells.3. PPAR-αmRNA expression in mouse liver cells infected bypAdEasy-HA-HCV core 3a adenovirus lower than pAdEasy-HA-HCVcore 1b and normal mouse hepatocytes. ACC1 expression wasincreased, while the expression of FAS did not change significantly. The AML12 was infected with pAdEasy-HA-HCV core 3a and 1b for24h. After extraction of total cellular RNA, reverse transcription, we usedreal-time quantitative PCR detect PPAR-α, ACC1 and FAS in the mRNAcontent. We found that infected with subtype 3a, PPAR-αexpression wassignificantly reduced. The ACC1 expression was increased, but there is only alittle difference between subtypes. The FAS mRNA expression hardlychanged.ConclusionThis study found that HCV core protein 3a is more strongly induced theliver steatosis than subtype 1b at the cellular level. Lipid droplets both in sizeand number may show this trend. PPAR-αplays a key role in steatosisinduced by HCV core protein.

Related Dissertations

CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Liver metabolic disorders
© 2012 www.DissertationTopic.Net Mobile