| Objective1.To explore the placental HBV infection and HBV intrauterine infection ofHBsAg positive pregnant women;2.To explore the gene level and the protein distribution of Toll like receptor3(TLR3) and nulear factor(NF-κB p65)in placental tissue of normal pregnant women and HBsAg positive pregnant women and its influencing factors;3.To explore the relationship between Toll like receptor3(TLR3) and nulear factor P65(NF-κB p65) in placental and maternal HBV infection and HBV intrauterine infection;4.To explore the role of TLR3, NF-κB P65,IFN-β in the placental of HBVinfection and HBV intrauterine infection;MethodSelect68cases of HBsAg positive pregnant women who were seized anddelivery at the Tai an central hospital from July2010to November2011, at the same time, select30cases of normal pregnant women, and two groups of pregnant women were have the normal liver function.According to the principle of informed consent in pregnant women, we collect the elbow vein blood of pregnant women before delivery, and collect the cord blood and placenta tissue asepticallyin the delivery time, we conduct the following research:1.The HBsAg, HBeAg in the maternal peripheral blood and cord blood weredetected by double antibody sandwich enzyme-linked immunosorbent assay(ELISA), the levels of HBVDNA in the peripheral blood and cord blood of HBsAg positive women were detected by fluorescence quantitative polymerase chainreaction (FQ-PCR),and the concentration of IFN-β in the serum of HBsAg positi ve group and normal group were detected by double antibody sandwich enzyme-linked immunosorbent assay(ELISA);2.The distribution of HBsAg in placentas of HBsAg positive pregnant women were detected by immunohistochemistry and the protein distribution of Toll like receptor3,nuclear factor-kappa Bp65in placentas of HBsAg positive pregnant women and normal pregnant women were detected by Immunohistochemistry;3.The gene expression of Toll like receptor3,nuclear factor-kappa Bp65inplacentas of HBsAg positive pregnant women and normal pregnant women weredetected Reverse transcription polymerase chain reaction.Results1.There were11cases of HBsAg positive, while16cases of HBVDNA positive inumbilical cord blood of68cases of HBsAg positive pregnant women, arbitary positive of HBsAg or HBVDNA are20cases, so the HBV intrauterine infection group is have20cases, while48cases in intrauterine uninfected group, andthe rate of total intrauterine was29.41%(20/68);There were38cases of HBsAgHBeAg double positive in maternal serum, and the intrauterine infection ratewas36.84%(14/38);2.The difference of intrauterine infection rate between HBVDNA positiveand HBVDNA negative in maternal was statistically significant(χ~2=24.178，P<0.001);3.We used immunohistochemical to detect the HBsAg in placental tissue of68cases of HBsAg positive pregnant women, and33cases were HBsAg positivein placental tissue, the positive rate was48.53%(33/68).Decidual cells infectionrate was48.53%(33/68), trophoblastic cells infection rate was42.65%(29/68), Villous meseachymal cells infection rate was30.88%(21/68), Villous capillary endothelial cells infection rate was13.24%(9/68). The differences of HBsAg expression in various types of cells of the placentas were statistically significant, the line×list chi-square test(χ~2=26.314,P<0.001). There were many brown positive signals distribution in each type of placenta cells, the HBsAg positive signals decreased gradually from the maternal side to fetal side of the placenta, and the positive signals distributed mostly in the cytoplasm, distributed occasionally in the nucleus;4.The findings of immunohistochemical showed that:The expression ofTLR3proteins were positive in the placental tissue of the30cases of normal pre gnant women, and the TLR3proteins was expressed in various types of cells of the placentas.56cases of pregnant women expressed TLR3proteins in placentatissue of68cases of HBsAg positive pregnant women, and the positive rate was82.35%(56/68), the differences of TLR3distribution in the various types of cellsof placenta tissue were statistically significant in the normal cases and HBsAgpositive cases(Decidual cells:χ~2=31.099,P<0.001,trophoblastic cells:χ~2=21.95,P<0.001,Villous meseachymal cells:χ~2=11.086,P<0.01,Villous capillary endothelial cells:χ~2=9.728,P<0.01);5.The expression of both TLR3and NF-κBp65protein were lower in placental of HBsAg positive group than that of normal control group,the difference was statistically significant (P<0.05,P<0.05), the expression of TLR3protein werelower in placental of HBV intrauterine infection group than that in HBV intrauterine uninfected group and normal control group, the differences were statistically significant(P<0.05,P<0.05),the expression of TLR3protein were lower in HBV intrauterine uninfected group than that of normal control group, but the difference was not statistically significant(P>0.05).The expression of NF-κBp65protein were lower in placental of HBV intrauterine infection group and HBV intrauterine uninfected group than that of normal control group, the differences were statistically significant (P<0.001,P<0.001), the expression of NF-κBp65protein was lower in placental of HBV intrauterine infection group than that of HBV intrauterine uninfected group(P<0.001).The expression of both TLR3and NF-κBp65in placental of maternal serum HBeAg positive group than that of HBeAg negative group, the difference was statistically significant(P<0.05,P<0.05);6.RT-PCR showed that the expression of both TLR3mRNA and NF-κBp65mRNA in placental of normal control group were stronger than that of HBsAgpositive group, the differences were statistically significant(P<0.001; P<0.05),the expression of TLR3mRNA and NF-κBp65mRNAin placental of HBV intrauterine infection group were lower than that of HBV intrauterine uninfectedgroup and the normal control group(P<0.001,P<0.001; P<0.05, P<0.05), theexpression of TLR3mRNA in placental of HBV intrauterine uninfected groupwas lower than that of normal control group(P<0.05),the expression of NF-κBp65mRNA in placental of HBV intrauterine uninfected group was not lower thanthat of normal control group(P>0.05).The expression of TLR3mRNA andNF-κp65mRNA in placental of maternal serum HBVDNA positive group were lower than HBVDNA negative group,but the difference was not statisticallysignificant(P>0.05, P>0.05);7.The serum level of IFN-β in HBV intrauterine infection group was lowerthan the HBV intrauterine group and the normal control group, the differenceswere statistically significant(P<0.001,P<0.01).Difference between HBV intrauterine uninfected group and the normal control group were significant(P<0.01);8.The expression of TLR3mRNA in placental and IFN-β levels in serum ofpregnant women have a significant positive correlation(r=0.732,P<0.01).The expression of NF-κBp65mRNA in placental and IFN-β levels in serum of pregnantwomen also have a significant positive correlation(r=0.876,P<0.01). Expressionof TLR3mRNA and NF-κBp65mRNA in placental have a significant positivecorrelation (r=0.698,P<0.01).Conclusion1.The HBV intrauterine infection rate was29.41%in maternal serum with the HBsAg positive. The HBV intrauterine infection rate was36.86%in maternalserum with both HBsAg positive and HBeAg positive;2.HBeAg positive and HBVDNA positive in the maternal serum are risk factors of HBV intrauterine infection, HBeAg regulated down the expressions of TLR3and NF-κBp65in placental resulting in the increased of HBV intrauterine infection rate;3.Various types of cells in placental could be infected by Hepatitis B virus,and the Hepatitis B virus infection is decreased from the maternal side to the fetal side in placental. The placental barrier might play a role in the blockade of Hepatitis B virus intrauterine infection;4.The TLR3proteins distributed in various types of cells of placental, and the TLR3proteins mainly expressed in the cytoplasm of the placental cells, the expression of TLR3protein is rased gradually from the maternal side to the fetalside. The NF-κBp65protein mainly expressed in placental trophoblast cells, cytoplasm of endothelial cells and a small amount of the nucleus;5.The expression of TLR3, NF-κBp65in placental and the IFN-β in maternal serum and the maternal HBV infection have a correlation, TLR3、 NF-κBp65and IFN-β are protection factors to the maternal HBV infection and the protection factors to the HBV intrauterine infection;6.The decline expression of TLR3and NF-κBp65in placental and the IFN- β in maternal serum of the HBV intrauterine infection group causing the body toresist the virus weakened.There expression descending possible commonly leadto intrauterine infection of hepatitis B virus in the whole cascade signaling pathways. |
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