Establishment And Application Of The Model For Anti-hepatitis B Virus Drug Evaluation In Primary Duck Fetal Hepatocytes

Hepatitis B is an infectious diseases that is very harmful and epidemic allover the world. However, no effective drug and therapeutic methods can curehepatitis B so far. It is difficult to establish the cell and/or animal models foranti-HBV study, because HBV has a narrow host range, which is limited to humansand chimpanzees. Obviously, lack of HBV infection model limited the developmentof anti-HBV drugs. Therefore, it is of great significance to establish a more effectivemodel for elucidating the pathogenesis of HBV infection and developing theanti-HBV innovative drug.In this study, we explored the isolation and primary culture method of duck fetalhepatocytes from the embryonated, un-hatched, duck eggs layed by theDHBV-infected Shaoxing ducks, and established the model to evaluate anti-HBVdrug with a SYBR green dye-based quantitative PCR assay in primary duck fetalhepatocytes. Meanwhile, the anti-HBV activities of three natural extracts weredetected to validate the practicability of the cell model established.1． Isolation and primary culture of duck fetal hepatocytesShaoxing duck embryo served as the experimental material.The ordinary PCRassay was employed to confirm congenital infection duck embryo.The selected duckembryo with duck hepatitis virus （DHBV） was used to isolate duck fetal hepatocytes.The conditions of isolation and primary culture of duck fetal hepaticytes wereoptimized as follows:（1） The comparative study of the different embryonic ageshowed that the18-22days-old duck embryos were more befitting.（2） Thecontrastive investigation of the different conditions of cell dissociation indicated thatthe hepatocytes treated with the concentration of0.25%of pancreatic enzyme for10min is the optimization condition.（3） The comparative research of the different centrifugal rotational speeds showed that the centrifugal rotational speed at3000-5000r is the optimal parameter.（4） The contrastive study of the different serumconcentrations indicated that the serum concentration of10%can meet the need ofthe cell culture.（5） The comparative investigation of the imported and domesticserums indicated that the domestic serum can meet the need of the duck fetalhepatocyte culture.（6） The contrastive research of the different ice bath timeindicated that the ice bath for15-20min is the optimal parameter.（7） Thecomparative study of the different mediums showed that the RPMI1640and DMEMmedium can meet the need of the duck fetal hepatocyte culture.（8） The contrastiveinvestigation of the different growth factors indicated that insulin and hydrocortisonesuccinate sodium together can improve the growth of duck hepatocytes.（9） Thecomparative research of the different cell inoculation density showed that the cellinoculation density at4-8×10⁵copies.mL^-1is the optimal parameter.. Eventually, theoptimized parameters of isolation and culture of duck fetal hepaticytes were determinedby the above-mentioned screening tests.2． Establishment of a quantitative PCR assay for detection of DHBV-DNAIn order to establish a rapid, accurate and specific SYBR green dye-basedfluorescence quantitative PCR assay for detection of DHBV-DNA in primary duckfetal hepatocytes, we designed a pair of specific primers spaning two gaps of DHBVgenome and cloned the PCR amplification products of DHBV genome into thePMD^-18T carrier. The plasmid containing DHBV genome was constructed and usedto create the standard curve for quantitative analysis of DHBV-DNA. The specificity,sensitivity and repeatability of the established method were respectively analyzed.The results showed that the specific and quantitative detection of DHBV DNA can becarried out by the established method. The assay has good linear relation in1.0×102^-1.0×108copies·μL^-1. Its amplification efficiency and correlation coefficientwas103.8%and0.995, respectively. The detective sensitivity of the assay is1.0×102copies·μL^-1and its repeatability coefficient was3.03%. Therefore, the fluorescencequantitative PCR method established is a specific and sensitive assay for detection of DHBV-DNA levels in primary duck fetal hepatocytes.3． Evaluation of Anti-DHBV effects of three extracts in duck hepatocytesThe anti-DHBV effects of three natural extracts were evaluated by thefluorescence quantitative PCR system established in primary duck fetal hepatocyes.Lamivudine was served as a positive control drug. The results showed that thedandelion extract reduced the intracellular DHBV-DNA levels in primary duck fetalhepatocyes. On the3rd day, dandelion extract at50^-100μg/ml had significantinhibitory effect on DHBV-DNA replication （P<0.01）. On the6th day, dandelionextract1^-100ug/ml reduced significantly DHBV-DNA levels （P<0.01）. Moreover,dandelion extract at100μg/ml afforded stronger effect than the positive control druglamivudine. Cichoric acid at25^-100ug/ml inhibited markedly DHBV-DNAreplication on the3rd day （P<0.01）. On the6th day, cichoric acid at1^-100ug/mlexhibited significant inhibition on DHBV-DNA replication （P<0.01）. Oleanolic acidat10^-100ug/ml reduced significantly DHBV-DNA levels on the3rd days （P<0.01）.On the6th day, oleanolic acid at1^-100ug/ml inhibited markedly the DHBV-DNAreplication in primary duck fetal hepatocyes.（P<0.01）.In summay, the study established a model to evaluate anti-HBV drug by theSYBR green dye-based quantitative PCR assay for detection of DHBV-DNA inprimary duck fetal hepatocytes. Furthermore, the anti-DHBV efects of three naturalextracts were evaluted using the duck fetal hepatocyte model established. Theestablishment of this model provides a powerful platform to clarify the pathogenicmechanism of hepatitis B and screen anti-HBV new drugs.