胎盘细胞凋亡在HBsAg阳性孕妇PBMC母—胎转运机制中的作用
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摘要
目的探讨PBMC母-胎转运与HBV宫内感染的关系,了解其作用的生物学基础；探讨胎盘细胞凋亡对胎盘通透性的影响,了解其与PBMC母-胎转运及新生儿HBV感染的关系；探讨PBMC作为HBV的载体,将孕妇体内HBV转运至胎儿体内导致HBV宫内感染的作用机制。
方法1.人群研究
连续收集2005年1月至2009年2月间太原市区各省市级医院筛检,并在太原市传染病院(太原市第三人民医院)妇产科进行产前检查并分娩的HBsAg阳性孕妇为研究队列,从产前检查追踪观察至分娩,排除孕期有其他感染性疾病或接受免疫治疗的孕妇,共收集HBsAg阳性孕妇及其新生儿共450对作为研究对象。收集孕妇孕期及产后新生儿的流行病学基线资料；采集孕妇分娩前肘静脉血及其新生儿出生24小时内注射乙肝疫苗和乙肝高效价免疫球蛋白(HBIG)前的股静脉血；无菌条件下采集孕妇足月分娩时胎盘组织。进行下述研究：
(1)采用等位基因特异性PCR(As-PCR)法检测HBsAg阳性孕妇及其新生儿PBMC中HUMGSTM1、ACE基因多态性,筛选母亲为GSTM1基因表达型(GSTM1+)和/或为ACE基因杂合子(ID型),且其新生儿为GSTM1基因缺失型(GSTM1-)和/或为ACE基因纯合子(纯合插入Ⅱ型或纯合缺失DD型)者作为信息病例对。将GSTM1和ACE基因作为母亲的特异性等位基因,从信息病例对的新生儿外周血PBMC中检测母亲基因,判定是否发生PBMC母一胎转运。
(2)采用酶联免疫吸附试验(ELISA)检测上述方法确定的信息病例对中孕妇及其新生儿外周血HBeAg、HBe-Ab、HBc-Ab及新生儿外周血HBsAg.
(3)采用荧光定量聚合酶联反应(Fluorescence Quantitative PCR, FQ-PCR)检测信息病例对中母儿外周血血清、PBMC中HBV DNA含量及PBMC HBV cccDNA含量。
(4)采用FQ-PCR检测新生儿PBMC中的母亲信息,用△Ct值(Ct目的基因/Ctβ-globin)表示PBMC母-胎转运量。
(5)采用免疫组织化学ABC法检测胎盘组织HBsAg。
(6)采用脱氧核苷酸末端转移酶(TdT)介导的核苷酸(dUTP)缺口末端标记法,原位末端标记技术(Tune1法)检测胎盘组织凋亡指数(AI),采用免疫组化SP法检测胎盘组织Caspase3蛋白表达,采用real-time RT-PCR检测胎盘组织Caspase3mRNA表达。
(7)采用巢式病例对照研究分析母-胎细胞转运及新生儿PBMC HBV感染复制及宫内感染的危险因素研究。
(8)采用免疫荧光双标技术检测PBMC涂片上HBsAg和GST,判断感染HBV的PBMC转运情况。
2.体外实验
(1)采用100μlHBV DNA含量为5.0×106(copies/ml)的病人血清感染PBMC,用CCK-8试剂盒检测不同共培养时间点12h、24h、48h和72h细胞的生长状况。将共培养的细胞清洗4次,用荧光定量PCR法检测PBMC和洗液中的HBV DNA的含量。
(2)在1μm孔径Transwell小室共培养模型中,用流式细胞技术(FACS)检测Transwell小室上室感染HBV的PBMC与Bewo的融合现象。再用8μm孔径Transwell小室共培养模型观察PBMC的迁移,用流式细胞技术(FACS)检测下室中标记有绿色荧光的PBMC。
(3)共培养时设立三个组：正常对照组(Bewo)、Bewo+HBV血清共培养组、Bewo+感染HBV的PBMC共培养组,在共培养0h、12h、24h、48h后分别收集各组Bewo细胞,通过流式细胞技术(FACS)检测Bewo的凋亡情况。
(4)用FQ-PCR法检测感染HBV的PBMC与Bewo共培养组不同时间点(0h、12h、24h、48h)的Bewo细胞caspases3mRNA的表达情况。
(5)用FQ-PCR法检测感染HBV的PBMC与Bewo共培养组下室中的PBMC的HBVDNA和HBV cccDNA含量。
3.统计分析
资料核实后输入数据库,采用SPSS16.0for windows软件进行统计分析。计数资料采用χ2-test,计算各指标阳性率、χ2值、OR值及其95%可信区间；计量资料采用t检验、方差分析及相关性分析。
结果1HBsAg阳性孕妇PBMC母-胎转运与孕妇HBV感染及新生儿HBV感染的关系
(1)对连续收集的450对HBsAg阳性孕妇及其新生儿的PBMC采用等位基因特异性PCR检测GSTM1、ACE基因多态性：检出86对母亲GSTM1基因型且其新生儿GSTM1基因型的信息病例对,84对母亲ID基因型且其新生儿II/DD基因型的信息病例对,合计将GSTM1、ACE基因作为母亲的特异性等位基因时,共检出155对信息病例对。
(2)孕妇HBsAg、HBeAg双阳性组△Ct值(PBMC转运Ct/β-globin Ct值)低于非双阳性组,但差异无统计学意义(t=0.81,P=0.42)；孕妇大、小三阳组△Ct值(PBMC转运Ct/β-globinCt值)均分别低于非大、小三阳组(t=1.42,P=0.16；t=0.02,P=0.98),但差异均无统计学意义。HBsAg阳性孕妇血清HBV DNA各组△Ct值(PBMC转运Ct/β-globinCt值)差别无统计学意义(F=0.37,P>0.05)。
(3) HBsAg阳性孕妇PBMC HBV DNA阳性组△Ct值(PBMC转运Ct/β-globin Ct值)低于阴性组,差异有统计学意义(t=6.42,P=0.00)；HBsAg阳性孕妇PBMC HBV cccDNA阳性组△Ct值(PBMC转运Ct/β-globin Ct值)高于阴性组,差异有统计学意义(t=2.13,P=0.04)。
(4)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿宫内感染的指标,则宫内感染组△Ct值(PBMC转运Ct/β-globin Ct值)高于非宫内感染组,但差异无统计学意义(t=0.34,P=0.73)。将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断新生儿宫内感染的指标,则宫内感染组ACt值(PBMC转运Ct/β-globin Ct值)低于非宫内感染组,差异有统计学意义(t’=6.25,P=0.00)。
2.胎盘HBV感染与HBsAg阳性孕妇母-胎细胞转运及新生儿HBV感染的关系
(1)胎盘HBV感染组△Ct值(PBMC转运Ct/β-globin Ct值)高于非感染组,但差异无统计学意义(t=0.38,P=0.70)；胎盘各型细胞HBV感染组ACt值(PBMC转运Ct/β-globin Ct值)除母体面的蜕膜细胞高于非感染组外其它均分别低于非感染组,但差异均无统计学意义(t=0.38,P=0.70；t=0.49,P=0.63；t=0.17,P=0.87；t=1.72,P=0.09)。
(2)胎盘HBV感染与新生儿血清HBsAg有关(χ2=4.88,P=0.03)而与新生儿HBV DNA无关(χ2=0.10,P=0.76)。将新生儿血清HBsAg、HBV DNA任一项阳性作为判断宫内感染的标准,胎盘HBV感染组的新生儿HBV宫内感染率高于非胎盘HBV感染组,但差异无统计学意义(χ2=2.71,P=0.10)。胎盘HBV感染与新生儿PBMC HBV DNA、PBMC HBV cccDNA均无关(χ2=1.89、0.76,P=0.17、0.38)。将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断宫内感染的标准,胎盘HBV感染组的新生儿HBV宫内感染率高于非胎盘HBV感染组,但差异无统计学意义(χ2=2.09,P=0.15)。
3.胎盘细胞凋亡与HBsAg阳性孕妇母-胎细胞转运及新生儿HBV感染的关系
(1)采用Tunel法检测155例HBsAg阳性孕妇胎盘的凋亡情况。胎盘细胞胞核出现棕黄色颗粒为凋亡阳性信号表达。从母体面至胎儿面的蜕膜细胞(DC)、滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)凋亡指数分别为4.5=3±0.07,5.56±0.06,4.66±0.05,4.05±0.06,合计胎盘细胞凋亡指数为4.75±0.05。凋亡指数在HBsAg阳性孕妇胎盘各层细胞上的分布总体上有统计学差异(F值为102.60,P=0.00),其中以滋养层细胞的凋亡为主,绒毛毛细血管内皮细胞的凋亡少见。采用SNK法进--步比较胎盘各层细胞凋亡指数,结果显示：蜕膜细胞(DC)凋亡指数与滋养层细胞(TC)、绒毛毛细血.管内皮细胞(VCEC)的凋亡指数差异均有统计学意义(P值均小于0.05),DC的凋亡指数低于TC而高于VCEC;TC与DC、VMC及VCEC的凋亡指数差异均有统计学意义,TC的凋亡指数高于DC、VMC和VCEC;DC的凋亡指数低于VMC的凋亡指数,但差异无统计学意义。
(2)发生PBMC母-胎转运组的胎盘细胞凋亡指数高于未发生转运组的胎盘细胞凋亡指数,差异有统计学意义(t=3.44,P=0.00)。发生PBMC母-胎转运组的胎盘各层细胞凋亡指数均高于未发生转运组,差异均有统计学意义(t=2.86,P=0.01；t’=4.86,P=0.00；t=2.53,P=0.01;t=2.23,P=0.03)。
(3)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞凋亡指数高于非宫内感染组,但差异无统计学意义(t=1.97,P=0.05)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞凋亡指数高于非宫内感染组,差异有统计学意义(t=3.22,P=0.00)。
(4)新生儿外周血PBMC HBV DNA阳性组的胎盘蜕膜细胞(DC)、滋养层细胞(TC)的凋亡指数高于阴性组,差异均有统计学意义(t=2.08,P=0.04；t=3.47,P=0.00)。绒毛间质细胞(VMC)、绒毛毛细血管内皮细胞(VCEC)的凋亡指数均高于阴性组,但差异无统计学意义(t’=0.92,P=0.36；t=0.77,P=0.44)。
(5)用免疫组织化学SP法检测155例HBsAg阳性孕妇胎盘Caspase3的表达,胎盘细胞细胞浆和/或细胞膜出现棕黄色染色为Caspase3阳性信号表达。从母体面的蜕膜细胞至胎儿面的蜕膜细胞(DC)、滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)Caspase3阳性表达的灰度值分别为98.40±1.86,82.05±1.39,86.41±1.57,89.10±1.70,合计胎盘Caspase3阳性表达的灰度值为88.99±1.47；积分光密度值分别为15.58±0.33,18.59±0.29,17.67±0.31,17.31±0.35,合计胎盘Caspase3阳性表达的积分光密度值为17.29±0.29。Caspase3蛋白在HBsAg阳性孕妇胎盘各层细胞上的分布总体上有统计学差异(F值分别为17.81、15.54,P值均小于0.05)。进一步采用SNK法进行两两比较,结果显示：蜕膜细胞(DC)Caspase3阳性表达的灰度值与滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)的灰度值差异均有统计学意义(P值均小于0.05),DC的Caspase3阳性表达灰度值均高于TC、VMC和VCEC, TC的Caspase3灰度值最小,即TC凋亡率最高,其次VMC、VCEC,DC凋亡率最低；TC与VMC、VMC与VCEC的Caspase3阳性表达的灰度值差异均无统计学意义。DC Caspase3阳性表达的积分光密度值与TC、VMC和VCEC的积分光密度值差异均有统计学意义,DC的Caspase3阳性表达积分光密度值均低于TC、VMC和VCEC; TC Caspase3阳性表达的积分光密度值与DC、VMC和VCEC的积分光密度值差异均有统计学意义,TC的Caspase3阳性表达积分光密度值均高于DC、VMC和VCEC,即TC凋亡率最高,其次VMC、VCEC,DC凋亡率最低：VMC与VCEC的Caspase3阳性表达的灰度值差异均无统计学意义。
(6)发生PBMC母-胎转运组胎盘细胞Caspase3IHC灰度值低于未发生转运组的灰度值,差异有统计学意义(t=2.80,P=0.01)；发生PBMC母-胎转运组胎盘细胞Caspase3IHC积分光密度值高于未发生转运组,差异有统计学意义(t=3.11,P=0.00)。发生PBMC母-胎转运组的胎盘各层细胞Caspase3IHC灰度值均低于未发生转运组,差异均有统计学意义(t=2.99,P=0.00；t=2.75,P=0.01；t=2.04,P=0.04;t=2.23,P=0.03)；发生PBMC母-胎转运组的胎盘各层细胞Caspase3IHC积分光密度值均高于未发生转运组,差异均有统计学意义(t=2.90,P=0.00；t=2.95,P=0.00;t=2.72,P=0.01；t=2.65,P=0.01)。
(7)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞Caspase3IHC灰度值低于非宫内感染组,积分光密度值高于非宫内感染组,但差异均无统计学意义(t=1.02,P=0.31；t=1.04,P=0.30)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞Caspase3IHC灰度值低于非宫内感染组,但差异无统计学意义(t=1.87,P=0.06)；积分光密度值高于非宫内感染组,差异有统计学意义(t=2.20,P=0.03)。
(8)新生儿外周血PBMC HBV DNA阳性组的胎盘蜕膜细胞(DC)、绒毛间质细胞(VMC)及绒毛毛细血管内皮细胞(VCEC)的Caspase3IHC灰度值均低于阴性组,积分光密度值均高于阴性组,但差异无统计学意义(t=1.55,P=0.12；t=1.48,P=0.14;t=1.18,P=0.24；t=1.62,P=0.11；t=0.67,P=0.50；t=0.89,P=0.38)；新生儿PBMC HBV DNA阳性组的胎盘滋养层细胞(TC)的Caspase31HC灰度值低于阴性组,积分光密度值高于阴性组,差异有统计学意义(t’=2.71,P=0.01；t=2.33,P=0.02)。
(9)胎盘组织Caspase3mRNAACt值(CtCaspase3/CtGAPDH值)与胎盘组织凋亡指数(AI)、Caspase3免疫组化积分光密度值(iod)均呈负相关(r=-0.66,P=0.00；r=-0.18,P=0.03),与胎盘组织Caspase3免疫组化灰度值(gray)量止相关(r=0.23,P=0.00)。
(10)发生PBMC母-胎转运组胎盘组织Caspase3mRNA的△Ct值(CtCaspase3/CtGAPDH值)低于未发生转运组的△Ct值,差异有统计学意义(t=3.86,P=0.00)。
(11)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘组织Caspase3mRNA的ACt值(CtCaspase3/CtGAPDH值)低于非宫内感染组,但差异无统计学意义(t=0.45,P=0.66)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘组织Caspase3mRNA的△Ct值(CtCaspase3/CtGAPDH值)低于非宫内感染组,差异有统计学意义(t=2.46,P=0.02)。
4.母-胎细胞转运及新生儿HBV感染的危险因素研究
(1)母-胎细胞转运危险因素的分析：孕妇PBMC HBV DNA、胎盘蜕膜细胞Caspase3蛋白水平及胎盘滋养层细胞凋亡指数被引入回归方程,OR值及95%CI分别为：14.60(5.61-38.00)、1.13(1.01-1.27)及4.09(1.898.85),且它们之间均存在交互作用(P值均<0.05,OR95%CI不包括1)。
(2)新生儿PBMC HBV DNA阳性危险因素的分析：孕妇PBMC HBV DNA、PBMC母-胎转运被引入回归方程,OR值及95%CI分别为：41.21(11.13-152.64)、17.09(4.91-59.56),且两因素间存在协同交互作用(P值<0.05,OR95%CI不包括1)。
(3)新生儿PBMC HBV cccDNA阳性危险因素的分析：孕妇PBMC HBV cccDNA阳性和胎盘凋亡指数被引入回归方程,OR值及95%CI分别为：40.87(9.63-173.41)和0.21(0.07-0.62),两因素间存在交互作用(P值<0.05,OR95%Cl不包括1)。
(4)新生儿HBV宫内感染危险因素的分析：将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇HBeAg阳性、分娩方式、胎盘滋养层细胞凋亡指数和孕妇年龄被引入回归方程,OR值及95%CI分别为：3.42(1.28-9.12)、0.33(0.12-0.95)、2.14(1.14-4.04)和0.87(0.77-0.99),交互作用分析显示除分娩方式和胎盘滋养层细胞凋亡指数两因素、胎盘滋养层细胞凋亡指数和孕妇年龄两因素间均无交互作用外(P值均>0.05,OR95%CI不包括1),其它两因素间均存在交互作用(P值均<0.05,OR95%CI不包括1)；将新生儿血清HBsAg、HBV DNA和／或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇PBMC HBV DNA阳性、PBMC母-胎转运、胎盘滋养层细胞凋亡指数被引入回归方程,OR值及95%CI分别为：6.95(2.71-17.82)、5.82(1.95-17.36)和2.56(1.33-4.91),交互作用分析显示它们之间均存在交互作用(P值均<0.05,OR95%CI不包括1)。
5.胎盘细胞凋亡在感染HBV的PBMC母-胎转运中的作用
(1)86例新生儿PBMC中检出20例HBsAg阳性者,阳性率为23.3%(20/86),即23.3%的新生儿发生PBMC HBV感染；31例GST阳性者,阳性率为36.0%(31/86),即36.0%的母儿发生了PBMC母-胎转运；HBsAg、GST共存者13例,阳性率为15.12%(13/86),即15.12%的母儿发生了感染HBV的PBMC转运;发生PBMC转运者中有41.94%(13/31)发生了感染HBV的PBMC转运。
(2)发生PBMC转运的新生儿发生PBMC HBsAg阳性的危险性是未发生PBMC转运的新生儿的4.952,有统计学意义(x2=9.477,P=0.002)。
(3)发生PBMC母-胎转运组的胎盘细胞凋亡指数高于未发生转运组的胎盘细胞凋亡指数,差异有统计学意义(t’=2.38,P=0.02)；且胎盘滋养层细胞的凋亡率高与PBMC转运相关(t=2.75,P=-0.01)。发生PBMC母-胎转运组的胎盘Caspase3蛋白的表达高于未发生转运组,但差异无统计学意义(t=0.23,P=0.82;t=0.98,P=0.33)。发生PBMC母-胎转运组的胎盘Caspase3mRNA的表达低于未发生转运组,但差异无统计学意义(t=1.59,P=0.12)。进一步分析胎盘细胞凋亡与感染HBV的PBMC转运的关系,但未显示感染HBV的PBMC转运与胎盘细胞凋亡率高及胎盘Caspase3蛋白和mRNA的表达有关。
(4)新生儿PBMC HBsAg阳性组的胎盘细胞凋亡指数低于阴性组的胎盘细胞凋亡指数,但差异无统计学意义(t=0.34,P=0.74)。新生儿PBMC HBsAg阳性组的胎盘Caspase3蛋白的表达略高于阴性组,但差异无统计学意义(t=0.01,P=0.99；t=0.07,P=0.95)。胎盘Caspase3mRNA的表达与新生儿PBMC HBsAg阳性亦无关(t=0.22,P=0.83)。
6.胎盘细胞凋亡在PBMC转运机制中作用的体外研究
(1)5×106copies/ml的HBV阳性血清刺激PBMC,共培养12h,24h和48hPBMC的细胞数不断增加,共培养72h左右,细胞数减少。与HBV血.清共培养48h的PBMC发生HBV感染,而清洗细胞的洗液中未检测出HBV DNA。
(2)在1μm孔径的Transwell小室共培养模型中Bewo细胞被染上了绿色荧光提示感染HBV的PBMC与Bewo发生了融合。以8μmn孔径的Transwell小室进行细胞迁移试验,下室中检测到标有绿色荧光的PBMC。
(3) Bewo与HBV血清和感染HBV的PBMC共培养0h、12h、24h、48h,Bewo+HBV感染组和Bewo+感染HBV的PBMC共培养组的早期凋亡率与对照组比较差异无统计学意义(P>0.05)。在共培养Oh、12h,Bewo+HBV感染组和Bewo+感染HBV的PBMC共培养组,Bewo总凋亡率与对照组比较差异无统计学意义(P>0.05);但共培养24h、48h,Bewo+HBV感染组总凋亡率与同期的对照组和Bewo+感染HBV的PBMC共培养组比较差异均有统计学意义(P<0.05)。分别对同一组内不同时间点的早期和晚期凋亡率进行比较,各组中不同时间点的凋亡率的差异有统计学意义(P<0.05),且随着共培养时间的延长,凋亡率呈现增加的趋势,到48h时HBV感染组总凋亡率达到最高。
(4)感染HBV的PBMC与Bewo共培养组不同的时间点Bewo细胞Caspase3mRNA的表达量比较差别有统计学意义(F=38.114,P=0.002),且各组间两两比较后,除12h与0hCaspase3mRNA的相对表达量无差异外,其他各组间差异均有统计学意义。在24h和48h组中,随着时间的延长Caspase3mRNA的相对表达量有升高的趋势。
(5)感染HBV的PBMC与Bewo细胞共培养48h时,收集下室的PBMC,其HBV DNA拷贝数为(2.565±0.361)×103copies/ml,同时,在此时间点HBV cccDNA的拷贝数为(1.3550±2.473)×103copies/ml,按判断标准HBV DNA拷贝数≥103copies/ml为阳性,HBVcccDNA拷贝数≥102copies/ml为阳性提示此时间点下室中的PBMC发生感染,且HBV发生复制。
结论1.将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断新生儿宫内感染的指标,即若将新生儿PBMC HBV DNA引入作为宫内感染的判定指标,PBMC母-胎转运与新生儿HBV宫内感染有关。
2.胎盘HBsAg阳性与新生儿血清HBsAg有关,而与宫内感染无关。
3.胎盘各层细胞均发生凋亡,以滋养层细胞凋亡为主。胎盘细胞凋亡增加与PBMC母-胎转运有关。将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,胎盘细胞凋亡与宫内感染无关。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,胎盘细胞凋亡则与宫内感染有关。滋养层细胞凋亡与新生儿PBMC HBV DNA有关。
4.孕妇PBMC HBV DNA、胎盘蜕膜细胞Caspase3蛋白表达及胎盘滋养层细胞凋亡指数是母-胎细胞转运的危险因素；孕妇PBMC HBV DNA、PBMC母-胎转运是新生儿PBMC HBVDNA阳性的危险因素；孕妇PBMC HBV cccDNA阳性是新生儿PBMC HBV cccDNA阳性的危险因素,胎盘凋亡指数是新生儿PBMC HBV cccDNA的保护因素；将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇HBeAg阳性和胎盘滋养层细胞凋亡指数是危险因素,而剖宫产和孕妇年龄是保护因素；将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇PBMC HBV DNA阳性、PBMC母-胎转运及胎盘滋养层细胞凋亡指数是危险因素。
5.以检测PBMC中HBsAg作为PBMC HBV感染的指标时,未显示感染HBV的PBMC转运与胎盘细胞凋亡有关。PBMC母-胎转运与新生儿PBMC HBsAg有关。胎盘细胞凋亡与新生儿PBMC HBsAg无关。
6.新生儿PBMC HBV DNA阳性可作为HBV宫内感染的诊断标准之一。PBMC可以作为HBV的载体从母体转运至胎儿血循环造成胎儿感染,PBMC母-胎转运可能是HBV宫内感染的另一条感染途径。
7.体外模拟胎盘屏障研究胎盘细胞凋亡和感染HBV的PBMC转运的关系,结果显示胎盘凋亡率与感染HBV的PBMC的迁移率正相关,且感染HBV的PBMC转运至Transwell下室后,可感染下室的PBMC,推论若感染HBV的母亲PBMC转运至胎儿血循环会造成胎儿PBMC HBV感染。
OBJECTIVE:To investigate the relationship between PBMC maternal-fetal transfer and HBV intrauterine infection and understand the biological basis of its role. To study the influence of placental apoptosis on placental permeability and understand the relationship between it and PBMC maternal-fetal transfer and neonatal HBV infection. To research the role mechanism of PBMC that is as HBV carrier and transport HBV from pregnant women into fetus and induce HBV intrauterine infection.
METHODS:1population studies:
HBsAg positive pregnant women and their newborns were consecutively collected from January (Jan.),2005to February (Feb.),2009in Taiyuan infectious hospital(third people's hospital of Taiyuan city). The above HBsAg positive pregnant women were selected by means of prenatal screening for HBsAg in provincial and municipal level hospitals of Taiyuan city and prenatal care and delivery in Obstetrics and Gynecology of Taiyuan infectious hospital. HBsAg positive pregnant women were continuously observed from prenatal examination to delivery. After pregnant women who had other infectious diseases or immune therapy were ruled out,450pairs HBsAg positive pregnant women and their newborns were collected as the research object. The epidemiology base line data involving gestation and postpartum were also collected, maternal elbow vein blood and femoral vein blood from newborns not only within24hours but also before infecting hepatitis B immuno-globulin (HBIG) were collected. At the same time, mature placenta were also collected under sterile conditions. Following studies were performed:
(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from HBsAg-positive pregnant women and their newborn. Mother-baby pairs informative cases for GSTM1would have the mother possessing the GSTM1gene and the baby possessing the null allele. And mother-baby pairs informative cases for ACE would have the mother possessing heterozygous ID gene type and the baby possessing homozygous insert Ⅱ genotype or homozygous deletion of DD genotype. In a word, informative cases for either GSTM1or ACE polymorphisms was subjected to As-PCR analysis for an allele which the mother possessed but which the baby did not. For the detection of the transfer of PBMC from the mother to the fetus,GSTM1or ACE allele were used as a maternal marker. PBMC maternal-fetal transfer was determined by detecting maternal marker in newborns' PBMC from informative cases.
(2) HBeAg,HBe-Ab,HBc-Ab in peripheral blood of HBsAg positive pregnant women who came from informative cases and HBsAg in peripheral blood of newborns who came from informative cases were determined by ELISA.
(3) HBV DNA level in serum and PBMC and HBV cccDNA level in PBMC in mothe-baby pairs informative cases were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR).
(4) Maternal marker was detected by FQ-PCR in PBMC of newborn from informative cases. ACt value(Cttarget gene/Ctβ-globin value) was used as showing the level of PBMC maternal-fetal transfer.
(5) HBsAg in placentas were detected by immunohistochemistry ABC method.
(6) TdT-mediated dUTP nick end labeling(TUNEL) method was used for informative cases placental samples to detect apoptosis index(AI). The expression of Caspase3in placenta was detected by a SP combination of immunohistochemistry techniques. Real time RT-PCR was used to detect the expression of Caspase3mRNA in placenta tissue.
(7) Risk factors of maternal-fetal cell transfer,HBV infection and duplication in PBMC and HBV intrauterine infection were analyzed by nested case-control studies.
(8) The expression of HBsAg and GST in PBMCs'glass slide of informative newborns were detected by the double immunofluorescent labeling method combined with confocal laser scanning microscope.
2. Experimental studies in vitro:
(1)100μl HBV DNA positive serum (the content of HBV DNA is5.0x106copies/ml) was added into PBMC in vitro for coculture. Then Cell Counting Kit-8was used to detect cell growth status at12h,24h,48h,72h. After coculture cells were washed four times by PBS,HBV DNA content in PBMC and lotion were detected by FQ-PCR.
(2) The fusion phenomenon between PBMC infected HBV and Bewo cells in the transwell upper chamber (24-mm-diameter and1μm pore size) was observed by fluorescence activated cells sorting(FACs). Then PBMC transfer was studied in the transwell chamber (24-mm-diameter and8μm pore size). PBMCs labeled with green fluorescent dye in the below chamber were detected by fluorescence activated cells sorting(FACs).
(3) This study had three groups:Bewo group(control group), HBV and Bewo coculture group, PBMC infected HBV and Bewo coculture group. They were respectively cultured for Oh,12h,24h,48h. FACs were used to detect apoptosis rate of Bewo cells at different time point of every group.
(4) Caspase3mRNA expression of Bewo cells was detected by Real-time RT-PCR at different time point (0h,12h,24h,48h) in PBMC infected HBV and Bewo coculture group.
(5) In PBMC infected HBV and Bewo coculture group FQ-PCR was used to detect HBV DNA and HBV cccDNA content of PBMCs in the below chamber.
3. Statistical analysis:Check and input the data, all of the data were analyzed by SPSS16.0for windows.χ2-test, t-test, ANOVAL and correlation analysis were used in the studies. RESULTS:1. The relationship between PBMC maternal-fetal transfer of HBsAg positive pregnant women and maternal HBV infection and neonatal HBV infection.
(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from450pairs ofHBsAg-positive pregnant women and their newborn. GSTM1genotyping revealed86informaitive cases in which the mother possessed an allele not present in the baby. ACE genotyping was then performed on the cases and revealed84informative cases. Thus, a total of155informative cases for the detection of maternal cells in fetal circulation were found uising these two systems.
(2) The level of PBMC maternal-fetal transfer was detected by FQ-PCR. There was no statistical differences between HBsAg, HBeAg both positive mothers and negative mothers. The same result emerged respectively in the comparison between Big Sanyang or Small Sanyang mothers and negative mothers. There was no statistical differences of the level of PBMC maternal-fetal transfer among four groups of serum HBV DNA content in mothers.
(3)ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV DNA positive group was lower than the negative group in HBsAg positive pregnant women, and the difference was statistically significant (t=6.42,P=0.00). ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV cccDNA positive was more than the negative group in HBsAg positive pregnant women, and the difference was also statistically significant (t=2.13,P=0.04).
(4) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group0=0.34, P=0.73). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group (t'=6.25,P=0.00)
2. The relationship between HBV infection in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.
(1) There was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV infection group and negative group in placenta (t=0.38, P=0.70). The same result was respectively showed among HBV infection group in placenta (t=0.38, P=0.70; t=0.49, P=0.63;t=0.17,P=0.87;t=1.72,P=0.09)
(2) HBV infection in placenta was associated with HBsAg in newborns'serum (χ2=4.88, P=0.03), but it was independent of HBV DNA in newborns'serum and PBMC HBV infection in placenta was respectively independent of HBV intrauterine infection and PBMC HBV cccDNA in newborns.
3. The relationship between cell apoptosis in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.
(1) The apoptosis index(AI) of155placental samples were detected by TdT-mediated dUTP nick end labeling(TUNEL) method. Placental cell nucleus appeared brown particle for expression of apoptosis positive signal. The Al was respectively4.53±0.07,5.56±0.06,4.66±0.05and4.05±0.06from the maternal side to the fetal side in the placental cell layers. The total of AI was4.75±0.05. Apoptotic index in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=102.60, P=0.00). The trophoblast cell apoptosis was higher than others, and villous capillary endothelial cell apoptosis was rare. Placental apoptosis index of each layer was compared with each other by using the analysis method of SNK. The results were that there were statistical significance between decidual cells and trophoblastic cells and villous capillary endothelial cells(P values less than0.05). But there were not statistical significance between decidual cells and villous mesenchymal cells.
(2) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.44, P=0.00). The same result was showed in the placental cell layers(t=2.86, P=0.01;t'=4.86, P=0.00; t=2.53, P=0.01; t=2.23, P=0.03).
(3) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=1.97, P=0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=3.22, P=0.00)
(4) Placental apoptosis index of PBMC HBV DNA positive group in newborns was more than the negative group in decidual cells and trophoblastic cells, and the difference was also statistically significant (t=2.08, P=0.04;t=3.47, P=0.00). But placental apoptosis was respectively independent of PBMC HBV DNA in newborns in villous mesenchymal cells and villous capillary endothelial cells.
(5) The expression of Caspase3in placentas were detected by immunohistochemistry SP method. Placental cell plasma and/or cell membrane appeared brown particle for expression of Caspase3positive signal. The gray value of Caspase3was respectively98.40±1.86,82.05±1.39,86.41±1.57and89.10±1.70from the maternal side to the fetal side in the placental cell layers. The total of gray value of Caspase3was88.99±1.47. The optical density of Caspase3was respectively15.58±0.33,18.59±0.29,17.67±0.31and17.31±0.35from the maternal side to the fetal side in the placental cell layers. The total of optical density of Caspase3was17.29±0.29. The expression of Caspase3in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=17.81,15.54, P value less than0.05). The expression of Caspase3of each layer was compared with each other by using the analysis method of SNK.The trophoblast cell apoptosis was higher than others, and decidual cells apoptosis was rare.
(6) The gray value of Caspase3of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=2.80, P=0.01), and the optical density of Caspase3of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.11, P=0.00). The same result was showed in the placental cell layers.
(7) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the expression of Caspase3between HBV intrauterine infection group and negative group(P>0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the optical density of Caspase3between HBV intrauterine infection group and negative group (t=2.20, P=0.03), but there was no statistical differences between both groups if the expression of Caspase3was showed by the gray value of Caspase3.
(8) The expression of Caspase3of PBMC HBV DNA positive group in newborns was more than the negative group in trophoblastic cells, and the difference was also statistically significant (t'=2.71, P=0.01; t=2.33, P=0.02). But the expression of Caspase3was respectively independent of PBMC HBV DNA in newborns in other placental cell layers.
(9) There was significantly negative correlation among Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) and the Al, optical density of Caspase3in placenta(r=-0.66, P=0.00; r=-0.18, P=0.03),and there was significantly positive correlation between Caspase3mRNA ΔCt value and gray value of Caspase3in placenta(r=0.23,P=0.00).
(10) Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=3.86, P=0.00).
(11) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=0.45, P=0.66). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=2.46, P=0.02)
4. Risk factors of PBMC maternal-fetal transfer and HBV infection in neonatal were studied.
(1) The risk factors of PBMC maternal-fetal transfer were analysis by logistic regression model. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells were all introduced to the regression equation. OR values and95%CI were respectively14.60(5.61-38.00),1.13(1.01-1.27) and4.09(1.89-8.85), and there were interaction among of them (P<0.05, OR95%Cl did not include one).
(2) The risk factors of PBMC HBV DNA positive in newborns were analysis by logistic regression model. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer were introduced to the regression equation. OR values and95%CI were respectively41.21(11.13-152.64) and17.09(4.91-59.56), and there was an synergistic interaction between the two factors (P<0.05, OR95%CI did not include one).
(3) The risk factors of PBMC HBV cccDNA positive in newborns were analysis by logistic regression model. PBMC HBV cccDNA in pregnant women and apoptosis index of placenta were introduced to the regression equation. OR values and95%CI were respectively40.87(9.63-173.41) and0.21(0.07-0.62), and there was an interaction both of them (P<0.05, OR95%CI did not include one).
(4) The risk factors of HBV intrauterine infection in newborns were analysis by logistic regression model. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women, mode of delivery, apoptosis index of placental trophoblast cells and age of pregnant women were introduced to the regression equation. OR values and95%CI were respectively3.42(1.28-9.12),0.33(0.12-0.95),2.14(1.14-4.04) and0.87(0.77-0.99). Interaction analysis showed that among apoptosis index of placental trophoblast cells and mode of delivery, age of pregnant women had no interaction, and among of other factors had interaction (P<0.05, OR95%CI did not include1). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells were introduced to the regression equation. OR values and95%CI were respectively6.95(2.71-17.82),5.82(1.95-17.36) and2.56(1.33-4.91), and there were interaction among of them (P<0.05, OR95%CI did not include one).
5. Role of placental apoptosis on maternal-fetal transfer of PBMC infected with HBV was studied.
(1) HBsAg positive rate was23.3%(20/86) in86cases of newborns'PBMC, that is23.3%of PBMC HBV infection in neonatal. The expression positive rate of GST was36.0%(31/86), that is36.0%of the mother-baby pairs occurring PBMC maternal-fetal transfer. There were13cases which showed coexistence of HBsAg and GST in the same PBMC of newborns, and the positive rate was15.12%(13/86), or15.12%of the mother-baby pairs occurring maternal-fetal transfer of PBMC infected with HBV. There were41.94%(13/31) of maternal-fetal transfer of PBMC infected with HBV in newborns who occurred PBMC maternal-fetal transfer.
(2) The risk of HBsAg positive in PBMC of newborns who occurred PBMC transportion was more than that of non transportion, and the risk multiple was4.952(χ2=9.477,P=0.002).
(3) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was statistically significant (t'=2.38, P=0.02). The higher rate of apoptosis in the trophoblast cells was closely related to PBMC maternal-fetal transfer(t=2.75, P=0.01). PBMC transfer from mother to baby was independent of the expression of Caspase3in placenta, and transfer of PBMC infected with HBV was also independent of the expression of Caspase3in placenta.
(4) There were no correlation among HBsAg positive in newborns'PBMC and placental apoptosis index, the expression of Caspase3in placenta(P>0.05).
6. Role of placental apoptosis on maternal-fetal transfer mechanism of PBMC was studied in vitro.
(1) The cell counts cultured at12h,24h and48h with positive serum were increasing,but declining at72h.HBV DNA can be detected in PBMCs co-cultured with HBV DNA positive serum for48hours and can't be detected in washing liquid after be washed four times by PBS
(2) A redistribution of the fluorescent dye was observed from the apical HBV-infected PBMCs to Bewo cells,indicating a fusion between HBV-infected PBMCs and Bewo cells in the Transwell model (1-mm porosity). In the Transwell model (8-mm porosity),PBMCs loaded with a fluorescent dye were detected in the basolateral chamber,which demonstrated that co-culture model in vitro to simulate transcytosis of the placental barrier was successfully established.
(3) When HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group were cultured for0h,12h,24h,48h. The difference of early apoptotic rates between HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group was not statistically significant with all the value of P more than0.05. However, while cocultured time was24h or48h, total apoptotic rates had a statistically significant difference in three groups(P<0.05). early apoptotic rates and total apoptotic rates were all statistically significant(P<0.05) compared to respectively control group at the time0h,12h,24h or48h.when the cocultured time was48h, total apoptotic rates were higher in the HBV and Bewo cells group.
(4) Compare caspase3mRNA of Bewo cells at different times in HBV+PBMCand Bewo cell groups. There was statistically significant difference (F=38.114,P=0.002). The relative expression of caspase3mRNA in48h group was higher than Oh、12h and24h group.(P<0.05),but there was also not significant difference between12h group and Oh group(P>0.05)
(5) HBV DNA and HBV cccDNA expression of PBMCs in the basolateral chamber were detected.HBV DNA content was (2.565±0.361)×103copies/ml and HBV cccDNA content was (1.3550±2.473)X103copies/ml.These results demonstrated that HBV can infected PBMCs and reproducted in them.
CONCLUSIONS:1. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no correlation between HBV intrauterine infection and PBMC maternal-fetal transfer. PBMC maternal-fetal transfer is correlated with PBMC HBV DNA in newborns. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, it is correlated with PBMC maternal-fetal transfer.
2. HBsAg positive in placenta is associated with HBsAg in newborns'serum. HBsAg positive in placenta is independent of HBV infection in newborns.
3. Placental layers of cells show apoptosis. The trophoblast cell apoptosis is higher than others. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no relation between placental apoptosis and HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was correlation between HBV intrauterine infection and placental apoptosis. PBMC HBV DNA positive in newborns is related with placental apoptosis in trophoblastic cells.
4. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells are risk factors for PBMC maternal-fetal transfer. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer are risk factors for PBMC HBV DNA positive in newborns. PBMC HBV DNA in pregnant women is a risk factor for PBMC HBV cccDNA positive in newborns, but apoptosis index of placenta is a protective factor. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection, but cesarean section and age of pregnant women are protective factors for HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection.
5. HBsAg in PBMC is as indicator of HBV infection in PBMC. Transfer of PBMC infected with HBV is independent of the placental apoptosis. PBMC HBsAg positive in newborns is related with PBMC transfer. There is no correlation among HBsAg positive in newborns'PBMC and placental apoptosis.
6. PBMC HBV DNA positive in newborns can be used as one of diagnostic criteria for HBV intrauterine infection. PBMC can be used as the carrier of the HBV which transport HBV from the mothers to their fetal blood circulation, and can cause fetal infection. PBMC maternal-fetal transfer may be another infection route of HBV intrauterine infection.
7. The relationship between placental apoptosis and transportion of PBMC infected with HBV was studied by imitating placental barrier in vitro. A positive correlation is found between the rate of placental apoptosis and the rate of PBMC transfer. After PBMCs infected with HBV come to below chamber of transwell through placental barrier, PBMCs below chamber can be infected. Thus, an inference come into being, that is PBMCs infected with HBV of mothers' transport to the fetal blood circulation and result in PBMC HBV infection in fetus.
方法1.人群研究
连续收集2005年1月至2009年2月间太原市区各省市级医院筛检,并在太原市传染病院(太原市第三人民医院)妇产科进行产前检查并分娩的HBsAg阳性孕妇为研究队列,从产前检查追踪观察至分娩,排除孕期有其他感染性疾病或接受免疫治疗的孕妇,共收集HBsAg阳性孕妇及其新生儿共450对作为研究对象。收集孕妇孕期及产后新生儿的流行病学基线资料；采集孕妇分娩前肘静脉血及其新生儿出生24小时内注射乙肝疫苗和乙肝高效价免疫球蛋白(HBIG)前的股静脉血；无菌条件下采集孕妇足月分娩时胎盘组织。进行下述研究：
(1)采用等位基因特异性PCR(As-PCR)法检测HBsAg阳性孕妇及其新生儿PBMC中HUMGSTM1、ACE基因多态性,筛选母亲为GSTM1基因表达型(GSTM1+)和/或为ACE基因杂合子(ID型),且其新生儿为GSTM1基因缺失型(GSTM1-)和/或为ACE基因纯合子(纯合插入Ⅱ型或纯合缺失DD型)者作为信息病例对。将GSTM1和ACE基因作为母亲的特异性等位基因,从信息病例对的新生儿外周血PBMC中检测母亲基因,判定是否发生PBMC母一胎转运。
(2)采用酶联免疫吸附试验(ELISA)检测上述方法确定的信息病例对中孕妇及其新生儿外周血HBeAg、HBe-Ab、HBc-Ab及新生儿外周血HBsAg.
(3)采用荧光定量聚合酶联反应(Fluorescence Quantitative PCR, FQ-PCR)检测信息病例对中母儿外周血血清、PBMC中HBV DNA含量及PBMC HBV cccDNA含量。
(4)采用FQ-PCR检测新生儿PBMC中的母亲信息,用△Ct值(Ct目的基因/Ctβ-globin)表示PBMC母-胎转运量。
(5)采用免疫组织化学ABC法检测胎盘组织HBsAg。
(6)采用脱氧核苷酸末端转移酶(TdT)介导的核苷酸(dUTP)缺口末端标记法,原位末端标记技术(Tune1法)检测胎盘组织凋亡指数(AI),采用免疫组化SP法检测胎盘组织Caspase3蛋白表达,采用real-time RT-PCR检测胎盘组织Caspase3mRNA表达。
(7)采用巢式病例对照研究分析母-胎细胞转运及新生儿PBMC HBV感染复制及宫内感染的危险因素研究。
(8)采用免疫荧光双标技术检测PBMC涂片上HBsAg和GST,判断感染HBV的PBMC转运情况。
2.体外实验
(1)采用100μlHBV DNA含量为5.0×106(copies/ml)的病人血清感染PBMC,用CCK-8试剂盒检测不同共培养时间点12h、24h、48h和72h细胞的生长状况。将共培养的细胞清洗4次,用荧光定量PCR法检测PBMC和洗液中的HBV DNA的含量。
(2)在1μm孔径Transwell小室共培养模型中,用流式细胞技术(FACS)检测Transwell小室上室感染HBV的PBMC与Bewo的融合现象。再用8μm孔径Transwell小室共培养模型观察PBMC的迁移,用流式细胞技术(FACS)检测下室中标记有绿色荧光的PBMC。
(3)共培养时设立三个组：正常对照组(Bewo)、Bewo+HBV血清共培养组、Bewo+感染HBV的PBMC共培养组,在共培养0h、12h、24h、48h后分别收集各组Bewo细胞,通过流式细胞技术(FACS)检测Bewo的凋亡情况。
(4)用FQ-PCR法检测感染HBV的PBMC与Bewo共培养组不同时间点(0h、12h、24h、48h)的Bewo细胞caspases3mRNA的表达情况。
(5)用FQ-PCR法检测感染HBV的PBMC与Bewo共培养组下室中的PBMC的HBVDNA和HBV cccDNA含量。
3.统计分析
资料核实后输入数据库,采用SPSS16.0for windows软件进行统计分析。计数资料采用χ2-test,计算各指标阳性率、χ2值、OR值及其95%可信区间；计量资料采用t检验、方差分析及相关性分析。
结果1HBsAg阳性孕妇PBMC母-胎转运与孕妇HBV感染及新生儿HBV感染的关系
(1)对连续收集的450对HBsAg阳性孕妇及其新生儿的PBMC采用等位基因特异性PCR检测GSTM1、ACE基因多态性：检出86对母亲GSTM1基因型且其新生儿GSTM1基因型的信息病例对,84对母亲ID基因型且其新生儿II/DD基因型的信息病例对,合计将GSTM1、ACE基因作为母亲的特异性等位基因时,共检出155对信息病例对。
(2)孕妇HBsAg、HBeAg双阳性组△Ct值(PBMC转运Ct/β-globin Ct值)低于非双阳性组,但差异无统计学意义(t=0.81,P=0.42)；孕妇大、小三阳组△Ct值(PBMC转运Ct/β-globinCt值)均分别低于非大、小三阳组(t=1.42,P=0.16；t=0.02,P=0.98),但差异均无统计学意义。HBsAg阳性孕妇血清HBV DNA各组△Ct值(PBMC转运Ct/β-globinCt值)差别无统计学意义(F=0.37,P>0.05)。
(3) HBsAg阳性孕妇PBMC HBV DNA阳性组△Ct值(PBMC转运Ct/β-globin Ct值)低于阴性组,差异有统计学意义(t=6.42,P=0.00)；HBsAg阳性孕妇PBMC HBV cccDNA阳性组△Ct值(PBMC转运Ct/β-globin Ct值)高于阴性组,差异有统计学意义(t=2.13,P=0.04)。
(4)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿宫内感染的指标,则宫内感染组△Ct值(PBMC转运Ct/β-globin Ct值)高于非宫内感染组,但差异无统计学意义(t=0.34,P=0.73)。将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断新生儿宫内感染的指标,则宫内感染组ACt值(PBMC转运Ct/β-globin Ct值)低于非宫内感染组,差异有统计学意义(t’=6.25,P=0.00)。
2.胎盘HBV感染与HBsAg阳性孕妇母-胎细胞转运及新生儿HBV感染的关系
(1)胎盘HBV感染组△Ct值(PBMC转运Ct/β-globin Ct值)高于非感染组,但差异无统计学意义(t=0.38,P=0.70)；胎盘各型细胞HBV感染组ACt值(PBMC转运Ct/β-globin Ct值)除母体面的蜕膜细胞高于非感染组外其它均分别低于非感染组,但差异均无统计学意义(t=0.38,P=0.70；t=0.49,P=0.63；t=0.17,P=0.87；t=1.72,P=0.09)。
(2)胎盘HBV感染与新生儿血清HBsAg有关(χ2=4.88,P=0.03)而与新生儿HBV DNA无关(χ2=0.10,P=0.76)。将新生儿血清HBsAg、HBV DNA任一项阳性作为判断宫内感染的标准,胎盘HBV感染组的新生儿HBV宫内感染率高于非胎盘HBV感染组,但差异无统计学意义(χ2=2.71,P=0.10)。胎盘HBV感染与新生儿PBMC HBV DNA、PBMC HBV cccDNA均无关(χ2=1.89、0.76,P=0.17、0.38)。将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断宫内感染的标准,胎盘HBV感染组的新生儿HBV宫内感染率高于非胎盘HBV感染组,但差异无统计学意义(χ2=2.09,P=0.15)。
3.胎盘细胞凋亡与HBsAg阳性孕妇母-胎细胞转运及新生儿HBV感染的关系
(1)采用Tunel法检测155例HBsAg阳性孕妇胎盘的凋亡情况。胎盘细胞胞核出现棕黄色颗粒为凋亡阳性信号表达。从母体面至胎儿面的蜕膜细胞(DC)、滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)凋亡指数分别为4.5=3±0.07,5.56±0.06,4.66±0.05,4.05±0.06,合计胎盘细胞凋亡指数为4.75±0.05。凋亡指数在HBsAg阳性孕妇胎盘各层细胞上的分布总体上有统计学差异(F值为102.60,P=0.00),其中以滋养层细胞的凋亡为主,绒毛毛细血管内皮细胞的凋亡少见。采用SNK法进--步比较胎盘各层细胞凋亡指数,结果显示：蜕膜细胞(DC)凋亡指数与滋养层细胞(TC)、绒毛毛细血.管内皮细胞(VCEC)的凋亡指数差异均有统计学意义(P值均小于0.05),DC的凋亡指数低于TC而高于VCEC;TC与DC、VMC及VCEC的凋亡指数差异均有统计学意义,TC的凋亡指数高于DC、VMC和VCEC;DC的凋亡指数低于VMC的凋亡指数,但差异无统计学意义。
(2)发生PBMC母-胎转运组的胎盘细胞凋亡指数高于未发生转运组的胎盘细胞凋亡指数,差异有统计学意义(t=3.44,P=0.00)。发生PBMC母-胎转运组的胎盘各层细胞凋亡指数均高于未发生转运组,差异均有统计学意义(t=2.86,P=0.01；t’=4.86,P=0.00；t=2.53,P=0.01;t=2.23,P=0.03)。
(3)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞凋亡指数高于非宫内感染组,但差异无统计学意义(t=1.97,P=0.05)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞凋亡指数高于非宫内感染组,差异有统计学意义(t=3.22,P=0.00)。
(4)新生儿外周血PBMC HBV DNA阳性组的胎盘蜕膜细胞(DC)、滋养层细胞(TC)的凋亡指数高于阴性组,差异均有统计学意义(t=2.08,P=0.04；t=3.47,P=0.00)。绒毛间质细胞(VMC)、绒毛毛细血管内皮细胞(VCEC)的凋亡指数均高于阴性组,但差异无统计学意义(t’=0.92,P=0.36；t=0.77,P=0.44)。
(5)用免疫组织化学SP法检测155例HBsAg阳性孕妇胎盘Caspase3的表达,胎盘细胞细胞浆和/或细胞膜出现棕黄色染色为Caspase3阳性信号表达。从母体面的蜕膜细胞至胎儿面的蜕膜细胞(DC)、滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)Caspase3阳性表达的灰度值分别为98.40±1.86,82.05±1.39,86.41±1.57,89.10±1.70,合计胎盘Caspase3阳性表达的灰度值为88.99±1.47；积分光密度值分别为15.58±0.33,18.59±0.29,17.67±0.31,17.31±0.35,合计胎盘Caspase3阳性表达的积分光密度值为17.29±0.29。Caspase3蛋白在HBsAg阳性孕妇胎盘各层细胞上的分布总体上有统计学差异(F值分别为17.81、15.54,P值均小于0.05)。进一步采用SNK法进行两两比较,结果显示：蜕膜细胞(DC)Caspase3阳性表达的灰度值与滋养层细胞(TC)、绒毛间质细胞(VMC)和绒毛毛细血管内皮细胞(VCEC)的灰度值差异均有统计学意义(P值均小于0.05),DC的Caspase3阳性表达灰度值均高于TC、VMC和VCEC, TC的Caspase3灰度值最小,即TC凋亡率最高,其次VMC、VCEC,DC凋亡率最低；TC与VMC、VMC与VCEC的Caspase3阳性表达的灰度值差异均无统计学意义。DC Caspase3阳性表达的积分光密度值与TC、VMC和VCEC的积分光密度值差异均有统计学意义,DC的Caspase3阳性表达积分光密度值均低于TC、VMC和VCEC; TC Caspase3阳性表达的积分光密度值与DC、VMC和VCEC的积分光密度值差异均有统计学意义,TC的Caspase3阳性表达积分光密度值均高于DC、VMC和VCEC,即TC凋亡率最高,其次VMC、VCEC,DC凋亡率最低：VMC与VCEC的Caspase3阳性表达的灰度值差异均无统计学意义。
(6)发生PBMC母-胎转运组胎盘细胞Caspase3IHC灰度值低于未发生转运组的灰度值,差异有统计学意义(t=2.80,P=0.01)；发生PBMC母-胎转运组胎盘细胞Caspase3IHC积分光密度值高于未发生转运组,差异有统计学意义(t=3.11,P=0.00)。发生PBMC母-胎转运组的胎盘各层细胞Caspase3IHC灰度值均低于未发生转运组,差异均有统计学意义(t=2.99,P=0.00；t=2.75,P=0.01；t=2.04,P=0.04;t=2.23,P=0.03)；发生PBMC母-胎转运组的胎盘各层细胞Caspase3IHC积分光密度值均高于未发生转运组,差异均有统计学意义(t=2.90,P=0.00；t=2.95,P=0.00;t=2.72,P=0.01；t=2.65,P=0.01)。
(7)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞Caspase3IHC灰度值低于非宫内感染组,积分光密度值高于非宫内感染组,但差异均无统计学意义(t=1.02,P=0.31；t=1.04,P=0.30)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘细胞Caspase3IHC灰度值低于非宫内感染组,但差异无统计学意义(t=1.87,P=0.06)；积分光密度值高于非宫内感染组,差异有统计学意义(t=2.20,P=0.03)。
(8)新生儿外周血PBMC HBV DNA阳性组的胎盘蜕膜细胞(DC)、绒毛间质细胞(VMC)及绒毛毛细血管内皮细胞(VCEC)的Caspase3IHC灰度值均低于阴性组,积分光密度值均高于阴性组,但差异无统计学意义(t=1.55,P=0.12；t=1.48,P=0.14;t=1.18,P=0.24；t=1.62,P=0.11；t=0.67,P=0.50；t=0.89,P=0.38)；新生儿PBMC HBV DNA阳性组的胎盘滋养层细胞(TC)的Caspase31HC灰度值低于阴性组,积分光密度值高于阴性组,差异有统计学意义(t’=2.71,P=0.01；t=2.33,P=0.02)。
(9)胎盘组织Caspase3mRNAACt值(CtCaspase3/CtGAPDH值)与胎盘组织凋亡指数(AI)、Caspase3免疫组化积分光密度值(iod)均呈负相关(r=-0.66,P=0.00；r=-0.18,P=0.03),与胎盘组织Caspase3免疫组化灰度值(gray)量止相关(r=0.23,P=0.00)。
(10)发生PBMC母-胎转运组胎盘组织Caspase3mRNA的△Ct值(CtCaspase3/CtGAPDH值)低于未发生转运组的△Ct值,差异有统计学意义(t=3.86,P=0.00)。
(11)将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘组织Caspase3mRNA的ACt值(CtCaspase3/CtGAPDH值)低于非宫内感染组,但差异无统计学意义(t=0.45,P=0.66)。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,宫内感染组的胎盘组织Caspase3mRNA的△Ct值(CtCaspase3/CtGAPDH值)低于非宫内感染组,差异有统计学意义(t=2.46,P=0.02)。
4.母-胎细胞转运及新生儿HBV感染的危险因素研究
(1)母-胎细胞转运危险因素的分析：孕妇PBMC HBV DNA、胎盘蜕膜细胞Caspase3蛋白水平及胎盘滋养层细胞凋亡指数被引入回归方程,OR值及95%CI分别为：14.60(5.61-38.00)、1.13(1.01-1.27)及4.09(1.898.85),且它们之间均存在交互作用(P值均<0.05,OR95%CI不包括1)。
(2)新生儿PBMC HBV DNA阳性危险因素的分析：孕妇PBMC HBV DNA、PBMC母-胎转运被引入回归方程,OR值及95%CI分别为：41.21(11.13-152.64)、17.09(4.91-59.56),且两因素间存在协同交互作用(P值<0.05,OR95%CI不包括1)。
(3)新生儿PBMC HBV cccDNA阳性危险因素的分析：孕妇PBMC HBV cccDNA阳性和胎盘凋亡指数被引入回归方程,OR值及95%CI分别为：40.87(9.63-173.41)和0.21(0.07-0.62),两因素间存在交互作用(P值<0.05,OR95%Cl不包括1)。
(4)新生儿HBV宫内感染危险因素的分析：将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇HBeAg阳性、分娩方式、胎盘滋养层细胞凋亡指数和孕妇年龄被引入回归方程,OR值及95%CI分别为：3.42(1.28-9.12)、0.33(0.12-0.95)、2.14(1.14-4.04)和0.87(0.77-0.99),交互作用分析显示除分娩方式和胎盘滋养层细胞凋亡指数两因素、胎盘滋养层细胞凋亡指数和孕妇年龄两因素间均无交互作用外(P值均>0.05,OR95%CI不包括1),其它两因素间均存在交互作用(P值均<0.05,OR95%CI不包括1)；将新生儿血清HBsAg、HBV DNA和／或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇PBMC HBV DNA阳性、PBMC母-胎转运、胎盘滋养层细胞凋亡指数被引入回归方程,OR值及95%CI分别为：6.95(2.71-17.82)、5.82(1.95-17.36)和2.56(1.33-4.91),交互作用分析显示它们之间均存在交互作用(P值均<0.05,OR95%CI不包括1)。
5.胎盘细胞凋亡在感染HBV的PBMC母-胎转运中的作用
(1)86例新生儿PBMC中检出20例HBsAg阳性者,阳性率为23.3%(20/86),即23.3%的新生儿发生PBMC HBV感染；31例GST阳性者,阳性率为36.0%(31/86),即36.0%的母儿发生了PBMC母-胎转运；HBsAg、GST共存者13例,阳性率为15.12%(13/86),即15.12%的母儿发生了感染HBV的PBMC转运;发生PBMC转运者中有41.94%(13/31)发生了感染HBV的PBMC转运。
(2)发生PBMC转运的新生儿发生PBMC HBsAg阳性的危险性是未发生PBMC转运的新生儿的4.952,有统计学意义(x2=9.477,P=0.002)。
(3)发生PBMC母-胎转运组的胎盘细胞凋亡指数高于未发生转运组的胎盘细胞凋亡指数,差异有统计学意义(t’=2.38,P=0.02)；且胎盘滋养层细胞的凋亡率高与PBMC转运相关(t=2.75,P=-0.01)。发生PBMC母-胎转运组的胎盘Caspase3蛋白的表达高于未发生转运组,但差异无统计学意义(t=0.23,P=0.82;t=0.98,P=0.33)。发生PBMC母-胎转运组的胎盘Caspase3mRNA的表达低于未发生转运组,但差异无统计学意义(t=1.59,P=0.12)。进一步分析胎盘细胞凋亡与感染HBV的PBMC转运的关系,但未显示感染HBV的PBMC转运与胎盘细胞凋亡率高及胎盘Caspase3蛋白和mRNA的表达有关。
(4)新生儿PBMC HBsAg阳性组的胎盘细胞凋亡指数低于阴性组的胎盘细胞凋亡指数,但差异无统计学意义(t=0.34,P=0.74)。新生儿PBMC HBsAg阳性组的胎盘Caspase3蛋白的表达略高于阴性组,但差异无统计学意义(t=0.01,P=0.99；t=0.07,P=0.95)。胎盘Caspase3mRNA的表达与新生儿PBMC HBsAg阳性亦无关(t=0.22,P=0.83)。
6.胎盘细胞凋亡在PBMC转运机制中作用的体外研究
(1)5×106copies/ml的HBV阳性血清刺激PBMC,共培养12h,24h和48hPBMC的细胞数不断增加,共培养72h左右,细胞数减少。与HBV血.清共培养48h的PBMC发生HBV感染,而清洗细胞的洗液中未检测出HBV DNA。
(2)在1μm孔径的Transwell小室共培养模型中Bewo细胞被染上了绿色荧光提示感染HBV的PBMC与Bewo发生了融合。以8μmn孔径的Transwell小室进行细胞迁移试验,下室中检测到标有绿色荧光的PBMC。
(3) Bewo与HBV血清和感染HBV的PBMC共培养0h、12h、24h、48h,Bewo+HBV感染组和Bewo+感染HBV的PBMC共培养组的早期凋亡率与对照组比较差异无统计学意义(P>0.05)。在共培养Oh、12h,Bewo+HBV感染组和Bewo+感染HBV的PBMC共培养组,Bewo总凋亡率与对照组比较差异无统计学意义(P>0.05);但共培养24h、48h,Bewo+HBV感染组总凋亡率与同期的对照组和Bewo+感染HBV的PBMC共培养组比较差异均有统计学意义(P<0.05)。分别对同一组内不同时间点的早期和晚期凋亡率进行比较,各组中不同时间点的凋亡率的差异有统计学意义(P<0.05),且随着共培养时间的延长,凋亡率呈现增加的趋势,到48h时HBV感染组总凋亡率达到最高。
(4)感染HBV的PBMC与Bewo共培养组不同的时间点Bewo细胞Caspase3mRNA的表达量比较差别有统计学意义(F=38.114,P=0.002),且各组间两两比较后,除12h与0hCaspase3mRNA的相对表达量无差异外,其他各组间差异均有统计学意义。在24h和48h组中,随着时间的延长Caspase3mRNA的相对表达量有升高的趋势。
(5)感染HBV的PBMC与Bewo细胞共培养48h时,收集下室的PBMC,其HBV DNA拷贝数为(2.565±0.361)×103copies/ml,同时,在此时间点HBV cccDNA的拷贝数为(1.3550±2.473)×103copies/ml,按判断标准HBV DNA拷贝数≥103copies/ml为阳性,HBVcccDNA拷贝数≥102copies/ml为阳性提示此时间点下室中的PBMC发生感染,且HBV发生复制。
结论1.将新生儿血清HBsAg、HBV DNA及PBMC HBV DNA任一项阳性作为判断新生儿宫内感染的指标,即若将新生儿PBMC HBV DNA引入作为宫内感染的判定指标,PBMC母-胎转运与新生儿HBV宫内感染有关。
2.胎盘HBsAg阳性与新生儿血清HBsAg有关,而与宫内感染无关。
3.胎盘各层细胞均发生凋亡,以滋养层细胞凋亡为主。胎盘细胞凋亡增加与PBMC母-胎转运有关。将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,胎盘细胞凋亡与宫内感染无关。将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准,胎盘细胞凋亡则与宫内感染有关。滋养层细胞凋亡与新生儿PBMC HBV DNA有关。
4.孕妇PBMC HBV DNA、胎盘蜕膜细胞Caspase3蛋白表达及胎盘滋养层细胞凋亡指数是母-胎细胞转运的危险因素；孕妇PBMC HBV DNA、PBMC母-胎转运是新生儿PBMC HBVDNA阳性的危险因素；孕妇PBMC HBV cccDNA阳性是新生儿PBMC HBV cccDNA阳性的危险因素,胎盘凋亡指数是新生儿PBMC HBV cccDNA的保护因素；将新生儿血清HBsAg、HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇HBeAg阳性和胎盘滋养层细胞凋亡指数是危险因素,而剖宫产和孕妇年龄是保护因素；将新生儿血清HBsAg、HBV DNA和/或PBMC HBV DNA任一项阳性作为判断新生儿HBV宫内感染的标准进行宫内感染危险因素的分析,孕妇PBMC HBV DNA阳性、PBMC母-胎转运及胎盘滋养层细胞凋亡指数是危险因素。
5.以检测PBMC中HBsAg作为PBMC HBV感染的指标时,未显示感染HBV的PBMC转运与胎盘细胞凋亡有关。PBMC母-胎转运与新生儿PBMC HBsAg有关。胎盘细胞凋亡与新生儿PBMC HBsAg无关。
6.新生儿PBMC HBV DNA阳性可作为HBV宫内感染的诊断标准之一。PBMC可以作为HBV的载体从母体转运至胎儿血循环造成胎儿感染,PBMC母-胎转运可能是HBV宫内感染的另一条感染途径。
7.体外模拟胎盘屏障研究胎盘细胞凋亡和感染HBV的PBMC转运的关系,结果显示胎盘凋亡率与感染HBV的PBMC的迁移率正相关,且感染HBV的PBMC转运至Transwell下室后,可感染下室的PBMC,推论若感染HBV的母亲PBMC转运至胎儿血循环会造成胎儿PBMC HBV感染。
OBJECTIVE:To investigate the relationship between PBMC maternal-fetal transfer and HBV intrauterine infection and understand the biological basis of its role. To study the influence of placental apoptosis on placental permeability and understand the relationship between it and PBMC maternal-fetal transfer and neonatal HBV infection. To research the role mechanism of PBMC that is as HBV carrier and transport HBV from pregnant women into fetus and induce HBV intrauterine infection.
METHODS:1population studies:
HBsAg positive pregnant women and their newborns were consecutively collected from January (Jan.),2005to February (Feb.),2009in Taiyuan infectious hospital(third people's hospital of Taiyuan city). The above HBsAg positive pregnant women were selected by means of prenatal screening for HBsAg in provincial and municipal level hospitals of Taiyuan city and prenatal care and delivery in Obstetrics and Gynecology of Taiyuan infectious hospital. HBsAg positive pregnant women were continuously observed from prenatal examination to delivery. After pregnant women who had other infectious diseases or immune therapy were ruled out,450pairs HBsAg positive pregnant women and their newborns were collected as the research object. The epidemiology base line data involving gestation and postpartum were also collected, maternal elbow vein blood and femoral vein blood from newborns not only within24hours but also before infecting hepatitis B immuno-globulin (HBIG) were collected. At the same time, mature placenta were also collected under sterile conditions. Following studies were performed:
(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from HBsAg-positive pregnant women and their newborn. Mother-baby pairs informative cases for GSTM1would have the mother possessing the GSTM1gene and the baby possessing the null allele. And mother-baby pairs informative cases for ACE would have the mother possessing heterozygous ID gene type and the baby possessing homozygous insert Ⅱ genotype or homozygous deletion of DD genotype. In a word, informative cases for either GSTM1or ACE polymorphisms was subjected to As-PCR analysis for an allele which the mother possessed but which the baby did not. For the detection of the transfer of PBMC from the mother to the fetus,GSTM1or ACE allele were used as a maternal marker. PBMC maternal-fetal transfer was determined by detecting maternal marker in newborns' PBMC from informative cases.
(2) HBeAg,HBe-Ab,HBc-Ab in peripheral blood of HBsAg positive pregnant women who came from informative cases and HBsAg in peripheral blood of newborns who came from informative cases were determined by ELISA.
(3) HBV DNA level in serum and PBMC and HBV cccDNA level in PBMC in mothe-baby pairs informative cases were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR).
(4) Maternal marker was detected by FQ-PCR in PBMC of newborn from informative cases. ACt value(Cttarget gene/Ctβ-globin value) was used as showing the level of PBMC maternal-fetal transfer.
(5) HBsAg in placentas were detected by immunohistochemistry ABC method.
(6) TdT-mediated dUTP nick end labeling(TUNEL) method was used for informative cases placental samples to detect apoptosis index(AI). The expression of Caspase3in placenta was detected by a SP combination of immunohistochemistry techniques. Real time RT-PCR was used to detect the expression of Caspase3mRNA in placenta tissue.
(7) Risk factors of maternal-fetal cell transfer,HBV infection and duplication in PBMC and HBV intrauterine infection were analyzed by nested case-control studies.
(8) The expression of HBsAg and GST in PBMCs'glass slide of informative newborns were detected by the double immunofluorescent labeling method combined with confocal laser scanning microscope.
2. Experimental studies in vitro:
(1)100μl HBV DNA positive serum (the content of HBV DNA is5.0x106copies/ml) was added into PBMC in vitro for coculture. Then Cell Counting Kit-8was used to detect cell growth status at12h,24h,48h,72h. After coculture cells were washed four times by PBS,HBV DNA content in PBMC and lotion were detected by FQ-PCR.
(2) The fusion phenomenon between PBMC infected HBV and Bewo cells in the transwell upper chamber (24-mm-diameter and1μm pore size) was observed by fluorescence activated cells sorting(FACs). Then PBMC transfer was studied in the transwell chamber (24-mm-diameter and8μm pore size). PBMCs labeled with green fluorescent dye in the below chamber were detected by fluorescence activated cells sorting(FACs).
(3) This study had three groups:Bewo group(control group), HBV and Bewo coculture group, PBMC infected HBV and Bewo coculture group. They were respectively cultured for Oh,12h,24h,48h. FACs were used to detect apoptosis rate of Bewo cells at different time point of every group.
(4) Caspase3mRNA expression of Bewo cells was detected by Real-time RT-PCR at different time point (0h,12h,24h,48h) in PBMC infected HBV and Bewo coculture group.
(5) In PBMC infected HBV and Bewo coculture group FQ-PCR was used to detect HBV DNA and HBV cccDNA content of PBMCs in the below chamber.
3. Statistical analysis:Check and input the data, all of the data were analyzed by SPSS16.0for windows.χ2-test, t-test, ANOVAL and correlation analysis were used in the studies. RESULTS:1. The relationship between PBMC maternal-fetal transfer of HBsAg positive pregnant women and maternal HBV infection and neonatal HBV infection.
(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from450pairs ofHBsAg-positive pregnant women and their newborn. GSTM1genotyping revealed86informaitive cases in which the mother possessed an allele not present in the baby. ACE genotyping was then performed on the cases and revealed84informative cases. Thus, a total of155informative cases for the detection of maternal cells in fetal circulation were found uising these two systems.
(2) The level of PBMC maternal-fetal transfer was detected by FQ-PCR. There was no statistical differences between HBsAg, HBeAg both positive mothers and negative mothers. The same result emerged respectively in the comparison between Big Sanyang or Small Sanyang mothers and negative mothers. There was no statistical differences of the level of PBMC maternal-fetal transfer among four groups of serum HBV DNA content in mothers.
(3)ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV DNA positive group was lower than the negative group in HBsAg positive pregnant women, and the difference was statistically significant (t=6.42,P=0.00). ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV cccDNA positive was more than the negative group in HBsAg positive pregnant women, and the difference was also statistically significant (t=2.13,P=0.04).
(4) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group0=0.34, P=0.73). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group (t'=6.25,P=0.00)
2. The relationship between HBV infection in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.
(1) There was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV infection group and negative group in placenta (t=0.38, P=0.70). The same result was respectively showed among HBV infection group in placenta (t=0.38, P=0.70; t=0.49, P=0.63;t=0.17,P=0.87;t=1.72,P=0.09)
(2) HBV infection in placenta was associated with HBsAg in newborns'serum (χ2=4.88, P=0.03), but it was independent of HBV DNA in newborns'serum and PBMC HBV infection in placenta was respectively independent of HBV intrauterine infection and PBMC HBV cccDNA in newborns.
3. The relationship between cell apoptosis in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.
(1) The apoptosis index(AI) of155placental samples were detected by TdT-mediated dUTP nick end labeling(TUNEL) method. Placental cell nucleus appeared brown particle for expression of apoptosis positive signal. The Al was respectively4.53±0.07,5.56±0.06,4.66±0.05and4.05±0.06from the maternal side to the fetal side in the placental cell layers. The total of AI was4.75±0.05. Apoptotic index in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=102.60, P=0.00). The trophoblast cell apoptosis was higher than others, and villous capillary endothelial cell apoptosis was rare. Placental apoptosis index of each layer was compared with each other by using the analysis method of SNK. The results were that there were statistical significance between decidual cells and trophoblastic cells and villous capillary endothelial cells(P values less than0.05). But there were not statistical significance between decidual cells and villous mesenchymal cells.
(2) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.44, P=0.00). The same result was showed in the placental cell layers(t=2.86, P=0.01;t'=4.86, P=0.00; t=2.53, P=0.01; t=2.23, P=0.03).
(3) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=1.97, P=0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=3.22, P=0.00)
(4) Placental apoptosis index of PBMC HBV DNA positive group in newborns was more than the negative group in decidual cells and trophoblastic cells, and the difference was also statistically significant (t=2.08, P=0.04;t=3.47, P=0.00). But placental apoptosis was respectively independent of PBMC HBV DNA in newborns in villous mesenchymal cells and villous capillary endothelial cells.
(5) The expression of Caspase3in placentas were detected by immunohistochemistry SP method. Placental cell plasma and/or cell membrane appeared brown particle for expression of Caspase3positive signal. The gray value of Caspase3was respectively98.40±1.86,82.05±1.39,86.41±1.57and89.10±1.70from the maternal side to the fetal side in the placental cell layers. The total of gray value of Caspase3was88.99±1.47. The optical density of Caspase3was respectively15.58±0.33,18.59±0.29,17.67±0.31and17.31±0.35from the maternal side to the fetal side in the placental cell layers. The total of optical density of Caspase3was17.29±0.29. The expression of Caspase3in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=17.81,15.54, P value less than0.05). The expression of Caspase3of each layer was compared with each other by using the analysis method of SNK.The trophoblast cell apoptosis was higher than others, and decidual cells apoptosis was rare.
(6) The gray value of Caspase3of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=2.80, P=0.01), and the optical density of Caspase3of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.11, P=0.00). The same result was showed in the placental cell layers.
(7) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the expression of Caspase3between HBV intrauterine infection group and negative group(P>0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the optical density of Caspase3between HBV intrauterine infection group and negative group (t=2.20, P=0.03), but there was no statistical differences between both groups if the expression of Caspase3was showed by the gray value of Caspase3.
(8) The expression of Caspase3of PBMC HBV DNA positive group in newborns was more than the negative group in trophoblastic cells, and the difference was also statistically significant (t'=2.71, P=0.01; t=2.33, P=0.02). But the expression of Caspase3was respectively independent of PBMC HBV DNA in newborns in other placental cell layers.
(9) There was significantly negative correlation among Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) and the Al, optical density of Caspase3in placenta(r=-0.66, P=0.00; r=-0.18, P=0.03),and there was significantly positive correlation between Caspase3mRNA ΔCt value and gray value of Caspase3in placenta(r=0.23,P=0.00).
(10) Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=3.86, P=0.00).
(11) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=0.45, P=0.66). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=2.46, P=0.02)
4. Risk factors of PBMC maternal-fetal transfer and HBV infection in neonatal were studied.
(1) The risk factors of PBMC maternal-fetal transfer were analysis by logistic regression model. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells were all introduced to the regression equation. OR values and95%CI were respectively14.60(5.61-38.00),1.13(1.01-1.27) and4.09(1.89-8.85), and there were interaction among of them (P<0.05, OR95%Cl did not include one).
(2) The risk factors of PBMC HBV DNA positive in newborns were analysis by logistic regression model. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer were introduced to the regression equation. OR values and95%CI were respectively41.21(11.13-152.64) and17.09(4.91-59.56), and there was an synergistic interaction between the two factors (P<0.05, OR95%CI did not include one).
(3) The risk factors of PBMC HBV cccDNA positive in newborns were analysis by logistic regression model. PBMC HBV cccDNA in pregnant women and apoptosis index of placenta were introduced to the regression equation. OR values and95%CI were respectively40.87(9.63-173.41) and0.21(0.07-0.62), and there was an interaction both of them (P<0.05, OR95%CI did not include one).
(4) The risk factors of HBV intrauterine infection in newborns were analysis by logistic regression model. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women, mode of delivery, apoptosis index of placental trophoblast cells and age of pregnant women were introduced to the regression equation. OR values and95%CI were respectively3.42(1.28-9.12),0.33(0.12-0.95),2.14(1.14-4.04) and0.87(0.77-0.99). Interaction analysis showed that among apoptosis index of placental trophoblast cells and mode of delivery, age of pregnant women had no interaction, and among of other factors had interaction (P<0.05, OR95%CI did not include1). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells were introduced to the regression equation. OR values and95%CI were respectively6.95(2.71-17.82),5.82(1.95-17.36) and2.56(1.33-4.91), and there were interaction among of them (P<0.05, OR95%CI did not include one).
5. Role of placental apoptosis on maternal-fetal transfer of PBMC infected with HBV was studied.
(1) HBsAg positive rate was23.3%(20/86) in86cases of newborns'PBMC, that is23.3%of PBMC HBV infection in neonatal. The expression positive rate of GST was36.0%(31/86), that is36.0%of the mother-baby pairs occurring PBMC maternal-fetal transfer. There were13cases which showed coexistence of HBsAg and GST in the same PBMC of newborns, and the positive rate was15.12%(13/86), or15.12%of the mother-baby pairs occurring maternal-fetal transfer of PBMC infected with HBV. There were41.94%(13/31) of maternal-fetal transfer of PBMC infected with HBV in newborns who occurred PBMC maternal-fetal transfer.
(2) The risk of HBsAg positive in PBMC of newborns who occurred PBMC transportion was more than that of non transportion, and the risk multiple was4.952(χ2=9.477,P=0.002).
(3) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was statistically significant (t'=2.38, P=0.02). The higher rate of apoptosis in the trophoblast cells was closely related to PBMC maternal-fetal transfer(t=2.75, P=0.01). PBMC transfer from mother to baby was independent of the expression of Caspase3in placenta, and transfer of PBMC infected with HBV was also independent of the expression of Caspase3in placenta.
(4) There were no correlation among HBsAg positive in newborns'PBMC and placental apoptosis index, the expression of Caspase3in placenta(P>0.05).
6. Role of placental apoptosis on maternal-fetal transfer mechanism of PBMC was studied in vitro.
(1) The cell counts cultured at12h,24h and48h with positive serum were increasing,but declining at72h.HBV DNA can be detected in PBMCs co-cultured with HBV DNA positive serum for48hours and can't be detected in washing liquid after be washed four times by PBS
(2) A redistribution of the fluorescent dye was observed from the apical HBV-infected PBMCs to Bewo cells,indicating a fusion between HBV-infected PBMCs and Bewo cells in the Transwell model (1-mm porosity). In the Transwell model (8-mm porosity),PBMCs loaded with a fluorescent dye were detected in the basolateral chamber,which demonstrated that co-culture model in vitro to simulate transcytosis of the placental barrier was successfully established.
(3) When HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group were cultured for0h,12h,24h,48h. The difference of early apoptotic rates between HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group was not statistically significant with all the value of P more than0.05. However, while cocultured time was24h or48h, total apoptotic rates had a statistically significant difference in three groups(P<0.05). early apoptotic rates and total apoptotic rates were all statistically significant(P<0.05) compared to respectively control group at the time0h,12h,24h or48h.when the cocultured time was48h, total apoptotic rates were higher in the HBV and Bewo cells group.
(4) Compare caspase3mRNA of Bewo cells at different times in HBV+PBMCand Bewo cell groups. There was statistically significant difference (F=38.114,P=0.002). The relative expression of caspase3mRNA in48h group was higher than Oh、12h and24h group.(P<0.05),but there was also not significant difference between12h group and Oh group(P>0.05)
(5) HBV DNA and HBV cccDNA expression of PBMCs in the basolateral chamber were detected.HBV DNA content was (2.565±0.361)×103copies/ml and HBV cccDNA content was (1.3550±2.473)X103copies/ml.These results demonstrated that HBV can infected PBMCs and reproducted in them.
CONCLUSIONS:1. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no correlation between HBV intrauterine infection and PBMC maternal-fetal transfer. PBMC maternal-fetal transfer is correlated with PBMC HBV DNA in newborns. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, it is correlated with PBMC maternal-fetal transfer.
2. HBsAg positive in placenta is associated with HBsAg in newborns'serum. HBsAg positive in placenta is independent of HBV infection in newborns.
3. Placental layers of cells show apoptosis. The trophoblast cell apoptosis is higher than others. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no relation between placental apoptosis and HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was correlation between HBV intrauterine infection and placental apoptosis. PBMC HBV DNA positive in newborns is related with placental apoptosis in trophoblastic cells.
4. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells are risk factors for PBMC maternal-fetal transfer. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer are risk factors for PBMC HBV DNA positive in newborns. PBMC HBV DNA in pregnant women is a risk factor for PBMC HBV cccDNA positive in newborns, but apoptosis index of placenta is a protective factor. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection, but cesarean section and age of pregnant women are protective factors for HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection.
5. HBsAg in PBMC is as indicator of HBV infection in PBMC. Transfer of PBMC infected with HBV is independent of the placental apoptosis. PBMC HBsAg positive in newborns is related with PBMC transfer. There is no correlation among HBsAg positive in newborns'PBMC and placental apoptosis.
6. PBMC HBV DNA positive in newborns can be used as one of diagnostic criteria for HBV intrauterine infection. PBMC can be used as the carrier of the HBV which transport HBV from the mothers to their fetal blood circulation, and can cause fetal infection. PBMC maternal-fetal transfer may be another infection route of HBV intrauterine infection.
7. The relationship between placental apoptosis and transportion of PBMC infected with HBV was studied by imitating placental barrier in vitro. A positive correlation is found between the rate of placental apoptosis and the rate of PBMC transfer. After PBMCs infected with HBV come to below chamber of transwell through placental barrier, PBMCs below chamber can be infected. Thus, an inference come into being, that is PBMCs infected with HBV of mothers' transport to the fetal blood circulation and result in PBMC HBV infection in fetus.
引文
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