| OBJECTIVE:The aim is to study the role of toll like receptor 3 playing in HBV infection and HBV intrauterine infection,based in population and in vitro.The clues of the relationship between TLR3 and HBV infection and HBV intrauterine infection from normal and HBsAg-positive pregnant women at gene and protein level were made,and then a series of experiments in vitro were demonstrated.1.To understand the HBV infection state of Peripheral Blood Mononuclear Cells(PBMC) in HBsAg positive pregnant women and their newborns.2.To understand the distribution of Toll Like Receptor 3(TLR3) on PBMC.3.To explore the role of TLR3 in human HBV infection and HBV intrauterine infection.4.To study the anti-virus mechanism of TLR3.METHODS:1 population-based studies:①228 HBsAg positive pregnant women were selected from July,2004 to January,2006 in Taiyuan infectious hospital.Followed studies were performed from the time of their beginning antenatal examination to the time of their giving birth to newborns and their newborns were also selected.Newborns' femoral vein blood within 24 hours before injecting HBV vaccine and mother's elbow vein blood before delivery were collected.At the same time,the epidemiological base line data involving gestation and postpartum were also collected.HBsAg and TLR3 in PBMC were detected by double labeling immunofluorescent histochemistry assay.②28 HBsAg positive pregnant women and their newborns were selected from November, 2006 to April,2007 in Taiyuan infectious hospital,at the same period,26 normal pregnant women were selected in Maternal and Child Health hospital of Shanxi province as the control, Newborns' femoral vein blood within 24 hours before injecting HBV vaccine and mother's elbow vein blood were collected,TLR3 mRNA in PBMC of HBsAg positive pregnant women and normal pregnant women was detected by RT-PCR;TLR3 mRNA in HBsAg positive pregnant women and their newborns PBMC was detected by real-time RT-PCR.HBsAg and HBeAg in gravidas' and newborns' serum were tested by ELISA.HBV DNA in both serums was detected by Fluorescence Quantitative PCR(FQ-PCR).The concentration of IL-2,6 in both serums of HBsAg positive and normal pregnant women were analyzed by ELISA. 2.Studies in vitro:①26 normal pregnant women in the first affiliated hospital of Shanxi Medical University were selected from April,2007 to October,elbow vein blood and the epidemiological base line data were collected.PBMC were isolated to culture with 1640 which contains 10%FBS.2 groups were defined:HBV stimulus and non- stimulus in each objects,PBMC and culture supernatant were selected respectively at 2nd,4th,6th culture day,TLR3 mRNA in PBMC was detected with real-time RT-PCR,the protein of TLR3 was detected by FACs,IL-2,6,10,12, IFN-γand TNF-αin supernatant were analyzed by ELISA.②TLR3 were silenced by RNAi and activated using PolyI:C that is a ligand of TLR3,then PBMC were cultured with HBV for 6 days,PBMC and culture supematant were selected respectively at 2nd,4th,6th culture day,TLR3 mRNA in PBMC was detected with real-time RT-PCR,the protein of TLR3 was detected by FACs,HBV DNA in both supernatant and PBMC were detected by FQ-PCR.3.Statistical analysis:Check and input the data,all of the data were analyzed by SPSS 13.0 for windows.PBMC HBV infection was analyzed by nested case-control study,χ~2-test,t-test, ANOVAL and correlation analysis.RESULTS:1.The relationship between TLR3 protein of HBsAg positive pregnant women'PBMC and PBMC HBV infection.①HBsAg was mostly located in the cytoplasm,a part on the cell membrane.TLR3 was mostly located on the cell membrane,while the distribution density of TLR3 was different at different PBMC smears,even at the same smears;the distribution was also different in each scope.The thicker TLR3 was,the thinner HBsAg was at one cell.②64 HBsAg positive were detected on PBMC smears of 228 HBsAg positive pregnant women and positive rate was 28.07%(64/228);161 PBMC smears were detected TLR3 positive and the positive rate was 70.61%(161/228),the difference of TLR3's expression between PBMC HBV infection group and non-infection group was statisticaly significant(χ~2=30.944,P＜0.0001); 35 PBMC smears were detected HBsAg positive from 228 HBsAg positive(in serum) pregnant women's newborn infants and the positive rate was 15.35%(35/228);The expression of TLR3 on pregnant women's PBMC was relative with PBMC HBV intrauterine infection of newborn infants(χ~2=12.354,P＜0.0001).TLR3 was probably a protective factor of PBMC HBV infection of HBsAg positive(in serum) pregnant women(OR=0.181,OR95%CI=0.091-0.340) and was also a protective factor of PBMC HBV intrauterine infection of newborn infants (OR=0.279,OR95%CI=0.133-0.585).2.The relationship between TLR3 mRNA of pregnant women'PBMC and PBMC HBV infection.①The performed tests of RT-PCR showed that TLR3 mRNA were higher in HBsAg positive pregnant women group than that in the normal.(t=-8.997,P＜0.001).②The performed tests of real time RT-PCR showed that TLR3 mRNA were significantly higher in HBsAg positive pregnant women than that in the normal(t=9.14,P＜0.0001 ).The rate of TLR3Ct/β-actin Ct was not significant difference among DNA negative group,HBV DNA≤104copies/ml group and HBV DNA＞104copies/ml group(F= 0.468,P＞0.05 ),and there were not significant difference between HBsAg,HBeAg and HBcAb positive group and the group that no same time positive group that in HBsAg positive pregnant women(t=0.355,P=0.725). HBsAg positive pregnant women were divided by their newborns having HBV intrauterine infection or not,the rate of TLR3Ct/β-actin Ct was not significant difference between the two groups(t=-1.525,P=0.139).③Newborns were divided by their having HBV intrauterine infection or not,the rate of TLR3Ct/β-actin Ct increased in HBV intrauterine infection group(t=2.613,P=0.015).3.The relationship between TLR3 mRNA of pregnant women'PBMC and the concentration of IL-2,6.①The concentration of IL-2 in HBsAg positive pregnant women serum(32.95±9.93 pg/ml) was higher than the normal pregnant women's(27.65±2.86 pg/ml ),while IL-6 was opposite to IL-2,(52.35±22.09 pg/ml ) vs.(544.6±615.4 pg/ml )(t=-2.291,P =0.028;t=3.575,P=0.002),the value of Th1/Th2 in HBsAg positive pregnant women(0.22±0.25) decreased compared with the normal(0.71±0.27)(P＜0.05).②The concentration of IL-6(81.00±21.64 pg/ml) in serum of HBsAg positive pregnant women with their newborns having HBV intrauterine infection highly increased compared with not having HBV intrauterine infection women's(42.80±11.7 pg/ml),(P=0.014).The value of Th1/Th2((0.43±0.13) vs.(0.80±0.24)) significantly decreased in these two groups(P=0.004).③Correlation analysis showed that the rate of TLR3Ct/β-actin Ct was negatively correlated with the concentration of IL-2,(r=-0.56,P＜0.05),the concentration of IL-2 increased with the TLR3 mRNA increasing.4.The relationship between TLR3 of normal pregnant women'PBMC which were stimulated by HBV DNA and the concentration of cytokines①PBMC from normal pregnant women were cultured,TLR3 increased in the HBV stimulus group compared to the HBV non-stimulus group(t=-2.36,P=0.029),but TLR3 mRNA at 2nd,4th,6th day was not significant difference(F=0.045,P=0.965). ②The concentration of IL-2,TNF-αin supematant of normal pregnant women PBMC cultured with HBV stimulus increased compared to the HBV non-stimulus group(t=2.415, P=0.023;t=2.277,P=0.031).IL-6,10,12,IFN-γand Th1/Th2(IL-2/IL-6) lightly increased but no significance.Correlation analysis showed that the value of TLR3Ct/β-actin Ct was significantly correlated with the concentration of IL-10,IL-6,IFN-γ,r was -0.331(P=0.043), -0.46(P=0.014),-0.557(P=0.025),respectively,that was to say,IL-10,IL-6 and IFN-γwill rise with TLR3 mRNA increasing.IL-12 was significantly correlated with the concentration of IFN-γ,IL-2,IL-10,rwas 0.588(P=0.027),0.475(P=0.014),-0.752,(P=0.001).IL-10 was significantly correlated with the concentration of IFN-γ,r was-0.633(P=0.006).IL-6 was significantly correlated with the concentration of TNF-α,IFN-γ,r was -0.467(P=0.019),-0.507 (P=0.045).5.TLR3 was silenced by RNAi or activated by PolyI:C which influence the replication of HBV①The depressed rate of PBMC TLR3 mRNA by RNAi was 67.83%,there was a significant difference at TLR3 mRNA level,as well as TLR3 protein,among healthy blood donor group, with HBV stimulus group,RNAi group and PolyI:C stimulus group(F=5.33,4.89; P=0.018,0.031).②TLR3 was depressed by RNAi for 48 hours both in mRNA and protein levels,contrast to no treatments,TLR3 mRNA gradually raised after culturing with HBV for 6 days and to maximum at the 4th day.The rising trend of TLR3 protein was the same as TLR3 mRNA,but it was to maximum at the 5th day.③TLR3 was augmented with PolyI:C provoking for 24 hours contrast to no treatments, TLR3 mRNA was continuously raised after culturing with HBV for 6 days and to maximum at the 2nd day.Then it was down at the 3rd day.TLR3 protein decreased at the 1st day,then rose gradually.④PBMC from healthy blood donors were cultured with HBV for 6 days,HBV DNA were detected at the 4th day and 6th day,the HBV DNA copies in supernatant and PBMC were no difference at the 2nd,4th,and 6th day(F=1.24,1.83,P=0.489,0.397).⑤PBMC activated by PolyI:C were cultured with HBV for 6 days,HBV DNA were detected at the 4th day,the content of HBV DNA in supernatant and PBMC decreased with the extension of time(F=6.34,7.01,P=0.036,0.027).⑥PBMC with RNAi were cultured with HBV for 6 days and at the 2nd day and the 6th day, HBV DNA were detected.Moreover,the content of HBV DNA in PBMC were significantly increased with the time(F=4.67,P=0.042 ),but there was no statistical significance in supernatant (F=2.34,P=0.249).CONCLUSIONS:1.TLR3 was expressed in PBMC and the density distribution of TLR3 was different at different kinds of cells.The thicker the TLR3 was,the thinner the HBsAg was at one cell.2.TLR3 was relative with the HBV infection,TLR3 mRNA of PBMC increased from HBsAg positive pregnant women,but TLR3 protein of PBMC that were infected by HBV decreased.TLR3 was a protective factor of PBMC HBV infection.Newborns' PBMC HBV intrauterine infection was definded by Newborns' PBMC HBV infection.The higher expression of TLR3 on pregnant women's PBMC was a protective factor of PBMC HBV intrauterine infection of their newborns.Newborns' HBV intrauterine infection was definded by HBsAg or HBV DNA positive in Newborns' serum,TLR3 on pregnant women's PBMC had no relations to HBV intrauterine infection of their newborns,TLR3 on newborns was a protective factor of newborns HBV intrauterine infection.3.TLR3 was positively correlated with Th1 cytokines,TLR3 may participate in anti-virus by increasing the production of cytokines.4.The concentration of IL-2,TNF-αin supernatant of normal pregnant women PBMC cultured with HBV stimulus were more than no HBV stimulus group.IL-6,10,12,IFN-γand Th1/Th2(IL-2/IL-6) were lightly increased,that showed PBMC can predominantly promote Th1 immunological reaction against HBV infection.5.PBMC were cultured with HBV stimulus,TLR3 increased both in mRNA and protein levels.When TLR3 was depressed by RNAi both in mRNA and protein levels,the susceptibility of PBMC to HBV was up-regulation.Moreover,if TLR3 were augmented with PolyI:C provoking both in mRNA and protein levels,the susceptibility of PBMC to HBV was down-regulation,TLR3 may influence HBV replication.6.TLR3 can be exploited as effect target of adjuvanticity,HBV vaccine or HBV treatment medicine. |
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