The Influence Of Hepatitis B Virus On The Expression Of The Immune Associated Molecules In Hepatocyte Cell Lines

Objects1. To detect the expression pattern of the immune associated molecules in three common hepatocyte cell lines.2. To compare the diversity of the innate immune associated molecules expression in different hepatocyte cell lines which were influenced by HBV replication in hepatocyte cell lines.3. To search for the difference of the expression of IL-10 between HepG2 and HepG2.2.15 and it's possible mechanism.Methods1. RT-PCR was used to detect the expression profile of the immune associated molecules in different hepatocyte cell lines (HepG2, HepG2.2.15 and Huh7).2. The HBV replication plasmids which named pHBV1.3 and pBS-HBV1.3 were used to transfect into HepG2 and Huh7 respectively. Real-time PCR was used to detect the innate immune associated molecules expression profiles such as TLRs,RLRs. CMIA was used to detect the expression of HBsAg and HBeAg in the cell culture supernatants. The HBV DNA level in the culture supernatants after transfection was monitored by real-time PCR.3. PCR and sequencing were used to identify the IL-10 mRNA expression in HepG2.2.15; ELISA was used to confirm the protein of IL-10 presence in the supernatant of the culture. After anti-viral treatments and transfection with HBVsiRNA, siRNA of IL-10 to HepG2.2.15, real-time PCR was used to detect the copies of HBV DNA in supernatant and mRNA of IL-10 expression in cells, CMIA was used to detect the expression of HBsAg and HBeAg, Elisa was used to detect the level of IL-10 protein in supernatant.4. Real-time PCR and western blot were used to detect the transcription factors such as STAT1, MyD88 and NF-κB expression in HepG2.2.15 after various treatments.Results1. The results of RT-PCR showed that all of the three cell lines(HepG2, HepG2.2.15 and Huh7) could express a series of immune-associated molecules. Compared with HepG2, the expression of TGF-β1, IL-10, IL-10R, TNFR, HLA-A and MICB were up-regulated, but IL=7, IL-15, IL-12p35, IL-15R, HLA-B, RIG1, A20 and DUBA were down-regulated in HepG2.2.15 in which HBV replication stably. It's interesting that the expression of IL-10 could only be detected in HepG2.2.15 cell lines.2. The HBV replication plasmids displayed different replication capacities in HepG2 and Huh7 cell lines. pBS-HBV1.3 produced rather low antigen and viral DNA level than pHBV1.3, not only in HepG2 but also in Huh7 cells. However, different replication capacities of HBV had different contribution to the regulation of the expression of the innate immune-associated molecules.3. IL-10 was secreted and expressed by HepG2.2.15, however, transfection with pHBV1.3 plasmid couldn't induce the expression of IL-10 in HepG2. It indicated that the expression of IL-10 perhaps has correlation to the persistence replication but not transient expression of HBV.4. The IL-10 siRNA could reduce the expression of IL-10 in both protein and mRNA level in HepG2.2.15 cells but had no significant influence on HBV replication.5. HBVsiRNA could inhibit the antigens and viral DNA level in HepG2.2.15, but had no significant influence to the level of HBV DNA. However the protein expression of IL-10 was down-regulated in HepG2.2.15 treated with HBV siRNA.6. Lamivudine could reduce the expression of antigens and the replication of HBV DNA in HepG2.2.15, and down-regulate the expression of IL-10's protein and mRNA. However the other non-specificity anti-viral chemicals affected the HBV DNA level in HepG2.2.15 but had no statistics significance. Except for poly(IC) could up-regulate the IL-10 protein expression, the other treatments down-regulated IL-10 protein expression in different degree. However, there had no significant influence to the expression of NF-κB and MyD88, but affected the expression of STAT1.Conclusions1. The three hepatocyte cell lines (HepG2,HepG2.2.15 and Huh7)which we used in this study all could express a series of immune-associated molecules, and the expression pattert in different cell lines was difference, the main difference occurring between HepG2 and HepG2.2.15, which derived from HepG2 and could express HBV stably. The greatest difference between HepG2 and Huh7 was that TLR7 expression only in Huh7. However, the expression of IL-10 only was detected in HepG2.2.15 cell line.2. The replication of HBV in hepatocyte cell lines affected the expression of some innate immune-associated molecules. The replication capacity of pBS-HBV1.3 in hepatocyte cell lines was more low than that of pHBV1.3, and different replication level of HBV has difference influence to the expression pattern of innate immune-associated molecules.3. HepG2.2.15 indeed expressed IL-10 but the induction and regulation mechanisms are independent of NF-κB, MyD88, and HBV DNA. Alternatively, STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15, but it was not the unique pathway. Furthermore, the activation of TLR3 pathway in HepG2.2.15 might be participated in regulation of IL-10 expression in the cell line, which is independent of HBV DNA level,NF-κB and MyD88, the certain mechanism need further study.