The Expression Of Umbilical Cord Blood Pdcs TLR9, IRF7, IFN-α In Maternal Infected With HBV In Phase Of Immune Tolerance And The Effects Of Bushen Jiedu Decoction On Their Expression

The first part:To establish the method of culturing pDCs in vitro.Objective:To establish a culture method of plasmacytoid dendritic cells(pDCs) in vitro.Methods:Umbilical cord blood of healthy maternal (40mL) was collected and the mononuclear cells were separated from the umbilical cord blood. The mononuclear cells were cultured for7days with RPMI1640complete medium containing rh FIt3-Ligand (Flt3-L)(100ng/ml) and rh interleukin(IL)-3(10ng/mL). The medium was changed one half every2days. On the eighth day, CpG ODN(2μg/mL) was administrated to the cells for24h, and the attached cells and the supernatant were collected for flow cytometry analysis and IFN-alpha secretion detection.During the culture, the morphological changes of pDCs were observed using inverted phase contrast microscope.Results:Two hours post culture, the umbilical cord blood mononuclear cells showed a circular, flat morphology. Most of the cells adhered to the wall, cytoplasm extended, their volume increased, the cells became round, translucent, and there were scattered small colony when cultured for24h. When cultured for3-4days, cell volume continued increasing than before,most of them were round, part of the cells’surface showed small protrusions, and a small amount of cells were spindle, the tadpole shape, a star or other irregular shaped cells. In the medium, colony increased significantly than before. When cultured for5-8days, number of colony and the cells number in the colony gradually reduced, and cells round or with small protuberances gradually increased in the medium. The cells expressing CD123, BDCA-2, BDCA-4were considered pDCs detected by flow cytometry. Flow cytometry revealed that the proportion of pDCs increased during the culture period with1.08%before culturing,5.32%at day4, and reaching19.8%at day8.On the eighth day, the level of IFN-alpha in pDC culture supernatant was11302+1745.31pg/mL. Conclusion pDCs can be induced successfully by rh Flt3-L combined with IL-3from human umbilical cord blood mononuclear cells.The second part:The influence of HBV on the expressions of TLR9, IRF-7, IFN-alpha in umbilical cord blood pDCs of healthy maternal.Objective:To observe the influence of HBV on the expressions of TLR9, IRF-7, IFN-alpha in umbilical cord blood pDCs of healthy maternal.Methods:HepG2.2.15cells were cultured in vitro. When cultured for twelfth days, the culture supernatant of HepG2.2.15was collected, then HBV virus particles were ultra centrifugated. HBV-DNA concentration was detected by fluorescence probe PCR. On the seventh day, pDCs and different load HBV (1*105copies/mL,1*106copies/mL,1*107copies/mL and vacuity contrast) co cultured for24h, then each group were cultured for24h with CpG ODN2216at a final concentration of2ug/mL. Expressions of pDCs TLR9, lRF7-mRNA were detected by Real-Time PCR, the expressions of pDCs TLR9, IRF7protein were measured by western blot, and the levels of IFN-alpha were measured by ELISAResults:HBV-DNA load was1.8*109copies/mL detected by fluorescence probe PCR which meeting the requirements of the experiment. The influence of HBV on the expression of pDCs TLR9, IRF-7mRNA was in a manner of concentration dependent. The higher HBV-DNA load, the stronger inhibition effect on the expression of IRF7mRNA TLR9mRNA. When the final concentration of HBV-DNA was1*107copies/ml, TLR9mRNA, IRF7mRNA level was the lowest. The influence of HBV on the expressions of pDCs TLR9, IRF7protein were also concentration dependent. The higher HBV-DNA load, the stronger inhibition effect was detected on the expression of IRF7, TLR9protein. When the final concentration of HBV-DNA was1*107copies/ml, TLR9, IRF7protein expression level was the lowest. The influence of HBV on the level of IFN-alpha in the supernatant also showed a concentration dependent. The higher HBV-DNA load, the stronger inhibition effect was detected in pDCs culture supernatant. When the final concentration of HBV-DNA was1*107copies/ml, the minimum IFN-alpha levels were tested in culture supernatant.The third part:The expression of umbilical cord blood pDCs TLR9, IRF7, IFN-alpha in maternal infected with HBV in phase of immune tolerance and the effects of Bushen Jiedu decoction on their expressionObjective:To compare the changes of umbilical cord blood pDCs TLR9, IRF7, IFN-alpha between maternal infected with HBV in immune tolerance phase and healthy maternal, and the effects of Bushen Jiedu decoction on umbilical cord blood pDCs TLR9, IRF7, IFN-alpha on maternal infected with HBV in immune tolerance phase.Methods:10maternals infected with HBV in immune tolerance phase and10healthy controls in the first affiliated hospital of hunan university of Chinese medicine were enrolled in this study. Umbilical cord blood pDCs were cultured in vitro. At the same time, the plasma containing drug of Bushen recipe (liu wei di huang wan), Jiedu recipe (gan lu xiao du dan), Bushen Jiedu recipe (liu wei di huang wan and gan lu xiao du dan)was preparated. On the seventh day, the pDCs were divided into5groups to intervene:blank group, Bushen drug plasma group, Jiedu drug plasma group, Bushen Jiedu drug plasma group and blank plasma group. After48h cultured, the expressions of TLR9, IRF7-mRNA were detected by real-time PCR, TLR9, IRF7protein were detected by Western blot, IFN-alpha in supernatant were measured by ELISA.Results:The expression of umbilical cord blood pDCs TLR9, IRF7mRNA in maternals infected with HBV in immune tolerance phase decreased obviously compared with normal maternal, the difference was statistically significant. The level of HBV-DNA in maternal peripheral blood was negatively related to pDCs TLR9, IRF7mRNA expression level. The plasma containing drug of Bushen recipe and Bushen Jiedu recipe could improve the expression of pDCs TLR9IRF7mRNA in maternals infected with HBV in immune tolerance phase to a certain extent, especially in the Bushen Jiedu group. Jiedu recipe drug plasma could not improve the expression of pDCs TLR9, IRF7mRNA. The expression of pDCs TLR9, IRF7protein in maternals infected with HBV in immune tolerance phase decreased significantly compared wih normal maternal. The level of HBV-DNA in maternal peripheral blood was negatively related to pDCs TLR9, IRF7protein expression level. The plasma containing drug of Bushen recipe and Bushen Jiedu recipe could improve the expression of pDCs TLR9IRF7protein in maternals infected with HBV in immune tolerance phase to a certain extent, especially in the Bushen Jiedu group. Although Jiedu recipe drug plasma could increase the expression of pDCs TLR9, IRF7protein to a certain extent, but there was no significant difference compared to the blank plasma. Expression IFN-alpha in pDCs supernatant in maternals infected with HBV in immune tolerance phase decreased significantly compared with normal maternal. The load of HBV-DNA in peripheral blood was negatively related to level of IFN-alpha in pDCs culture supernatant. The plasma containing drug of Bushen recipe and Bushen Jiedu recipe could improve the level of IFN-alpha in pDCs culture supernatant in maternals infected with HBV in immune tolerance phase to a certain extent, especially in the Bushen Jiedu group. Jiedu recipe drug plasma could not improve the e levels of IFN-alpha in pDCs culture supernatant.Conclusion:1. pDCs can be induced successfully combined by rh Flt3-L+IL-3from human CBMC which is initial cell.2.The expressions of normal pDCs TLR9, IRF7mRNA and protein are decreased intervened by HBV in vitro, resulting in reduced secretion of downstream IFN-a. 3. The expressions of umbilical cord blood pDCs TLR9, IRF7and the secretion of INF-alpha in maternal infected with HBV in phase of immune tolerance decrease obviously compared with normal maternal. The decrease expressions of pDCs TLR9, IRF7and the reduced secretion of INF-alpha impaired pDCs function are one the important causes of HBV escaping the immune response and resulting in chronic infection.4. Bushen Jiedu decoction can better promote the expressions of umbilical cord blood pDCs TLR9, IRF7and the secretion of INF-alpha in maternal infected with HBV in phase of immune tolerance. Bushen Jiedu decoction is the basic way to immune regulation and the treatment of chronic hepatitis B.