The Effect Of Supernatant Of Macrophages Stimulated By HCV Core Protein On Human Hepatocytes Biological Characteristics

Objective: HCV(Hepatitis C virus) is the most important pathogens lead to liver diseases, the majority of these infections developed chronic infection, which may eventually lead to liver fibrosis, cirrhosis and liver cancer. And there is no effective vaccine to prevent the infection to date. Pegylated interferon –α combined ribavirin Lin is the best treatment of hepatitis C, but still useless for about 30%-50% of the HCV patients. HCV core protein is the first expression in infected liver cells, which can be detected in the serum of patients and infected target cells. HCV core protein is not only involved in assembly of virus particles, but also regulates function of the target cell through interacting with different proteins of target cells, including proliferation, cell growth and apoptosis.The innate immune response is the first defense of body to prevent the pathogens. Kupffer cell in liver play an important role in the innate immune response, they also involved in the body’s immune defenses act as settled macrophages which are a class of cells of highly heterogeneous. Being stimulated in tissue microenvironment, they can be differentiated into cell subsets including Classically activated M1 cell, alternatively activated M2 cell, tumor-associated macrophages with different functions. Our pre-researches have revealed that the macrophage, m-THP1, secreted pro- and anti-inflammatory cytokines after being stimulate by HCV core protein, by collecting cells which include L02 cells which come from healthy persons and hepatoma cells, Hep G2 and SMMC-7721 cell cultured with the supernatant of macrophages stimulated by HCV core protein, this paper lays an emphasis on the influence of supernatant of macrophages on the proliferation, migration and invasion of these cells. Meanwhile a preliminary research of the mechanism is also done. The study provides the experimental basis for the research of the effect of core protein on the activity of macrophages further more.Methods:1 Expression and construction of recombinant HCV core proteinHCV core gene fragment was obtained from full gene plasmid of JFH-1 strain(HCV2a) by RT-PCR with designed primers.The RT-PCR product was inserted into p MD18-T vector and sequenced. The prokaryotic expression plasmid p ET28 a were cut with the same enzymes, Bam HI and HindⅢ. Thus a recombinant prokaryotic expression plasmid p ET28a/HCV core was constructed. After being induced by IPTG, the protein was expressed. Then through purification, renaturation, and concentrate, the HCV core protein was identified by SDS-PAGE and Western-blot.2 Collection of the supernatant of macrophages stimulated by HCV core proteinHuman monocytic cell line THP1 was differented into macrophages m-THP1 stimulated by PMA. The supernatant of macrophages stimulated by HCV core protein was obtained and centrifuged 24 h hours later. The supernatant of macrophages stimulated by PBS and complete medium containing HCV core protein were used as the control group at the same time.3 Observation of the effect on biological characters of these cells after being cultivated with the supernatant of macrophagesL02 cell, Hep G2 and SMMC-7721 cell were cultivated by 20% and 50% supernatant(20%supernatant +80% complete medium, 50% supernatant +50% complete medium). 1) The influence of supernatant on proliferation, migration capacity of cells was detected by CCK8 assay, Wound healing and Transwell chamber assay.2) The m RNA levels of MMP9 and MMP2 in three cells were explored through Real-Time PCR. 3) The change of protein expressed by liver and hepatoma cell, ERK,JNK,p38,NF-κB65 was detected by Western blot4 Observation of the effect on the sensitivity to IFN-α of these cells after being cultivated with the supernatant of macrophagesL02, Hep G2 and SMCC7721 cells were cultivated in 96-well cell culture plates, and the medium were replaced by which containing IFN-α 1000U/100 ul after adherent. Different concentrations of cell culture supernatant were added to reach 200 ul an hour later. The change of proliferation of these cells was detected in 24, 48 h by CCK8 assay. Meanwhile, let IFN-α group, supernatant of m-THP1 stimulated by core protein, supernatant of m-THP1 stimulated by PBS + IFN-α be control groups, and supernatant of m-THP1 stimulated by core protein+ IFN-αexperimental groups.Results:1 A prokaryotic expression plasmid p ET28a/HCVcore is successfully constructed(containing two His-tag).there are about 575 bp by agarose gel electrophoresis after being digested by Bam HⅠand HindⅢ, the results of sequencing are consistent with which in Genbank.2 HCV core protein is expressed by prokaryotic and a single band of about 21 KD detected by SDS-PAGE and Western blot after purification is obtained.3 The supernatant of m-THP1 stimulated by core protein significantly promotes cell proliferation, migration and invasion. 1) 50% supernatant of m-THP1 cells stimulated by HCV core can promote the proliferation ability of L02, Hep G2 and SMMC-7721 apparently than supernatant of m-THP1 cells stimulated by PBS or complete medium containing 50% HCV core protein. 2) The migration ability of SMMC-7721 cell is promoted by 20% supernatant of m-THP1 cells stimulated by HCV core detected through Woud healing and Transwell assay compared to control group, while 50% supernatant of m-THP1 cells stimulated by HCV core increase the migration ability of L02 cell. And there is no significantly statistical difference in the result of Hep G2 cell.4 The inhibition of IFN-α on hepatocytes is interfered by the supernatant of m-THP1 stimulated by core protein: the cell proliferation can be inhibited by IFN-α compared to control groups, while the cell proliferation are promoted when cultured with supernatant of m-THP1 cells stimulated by HCV core and can produce an interference on the inhibition of IFN-α.5 The levels of MMP9 in SMMC-7721 cell cultivated with supernatant of m-THP1 cells stimulated by HCV core are significantly increased than which cultivated with supernatant of m-THP1 cells stimulated by PBS or complete medium containing HCV core protein. There is no significant change in the level of MMP2 or MMP9 of L02 cell and Hep G2 cell compared to control group.6 Weatern-blot analysis indicates that the expression of p-ERK in SMMC-7721 cell is reduced when being cultivated with supernatant of m-THP1 cells stimulated by HCV core, while the expression of NF-κBp65 is increased, the expression of p-38 can not be affected by HCV core.Conclouion:1 Recombinant plasmid of HCV core protein which contains two his-tag was constructed and expressed.2 The proliferation ability of L02 cell, Hep G2 cell and SMMC-7721 cell can be promoted by supernatant of m-THP1 cells stimulated by HCV core. HCV core protein interact with macrophages can increase the migration ability of L02 cell and SMMC-7721 cell, which has a certain correlation with the existence of NF-κB.3 The sensitivity to IFN-α of L02 cell, Hep G2 cell and SMMC-7721 cell can be reduced when being cultivated with supernatant of m-THP1 cells stimulated by HCV core.