ICP母胎胆汁酸变化及胎盘BSEP、AE2表达的研究
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摘要
目的探讨胆盐载体BSEP、AE2在妊娠各期胎盘和妊娠期肝内胆汁淤积症(ICP)胎盘中的表达量，及其与母胎血中总胆汁酸和九种胆汁酸水平的关系，进一步分析胎盘胆盐载体BSEP、AE2在ICP病理机制中的作用。
方法收集妊娠晚期ICP患者(ICP组)及正常晚孕妇女(对照组)胎盘组织及母体静脉血、脐静脉血各20例；收集正常早、中孕胎盘组织各8例、7例。采用速率法检测母、脐静脉血中总胆汁酸(TBA)的含量；采用高效液相色谱质谱联用法检测两组母、脐静脉血中九种胆汁酸：胆酸(CA)，脱氧胆酸(DCA)，鹅脱氧胆酸(CDCA)，牛磺胆酸(TCA)，牛磺脱氧胆酸(TDCA)，牛磺鹅脱氧胆酸(TCDCA)，甘胆酸(GCA)，甘氨鹅脱氧胆酸(GCDCA)，石胆酸(LCA)水平；采用实时荧光定量PCR法测定胎盘组织中BSEP和AE2 mRNA的表达量；采用免疫组织化学法检测AE2在胎盘组织中的细胞学定位。
结果(1)ICP组母血和脐血中TBA水平均高于对照组(46．15±62．82 VS 2．82±1．82μmol／L；18．23±16．77μmol／L VS 4．79±1．26)：ICP组母血中TBA水平高于脐血(46．15±62．82 VS 18．23±16．77μmol／L)，对照组脐血TBA水平高于母血(4．79±1．26 VS 2．82±1．82μmol／L)，以上差异均有统计学意义(P＜0．05)。(2)ICP组母血中TCA、CA、TDCA、GCA、GCDCA、TCDCA水平高于对照组，DCA水平低于对照组；ICP组脐血中TCA、CA、TDCA、GCA、GCDCA、LCA水平高于对照组；ICP组母血中TCA、DCA、TDCA、TCDCA、GCA高于脐血；对照组母血中DCA、TDCA、LCA高于脐血，TCDCA低于脐血；ICP组母血九种胆汁酸成分与脐血相应成分之间均存在正相关(r=0．478～0．952)，对照组母血GCA、DCA、LCA与脐血相应成分之间存在正相关(r分别为0．585、0．764、0．595)，以上差异及相关性均有统计学意义(P＜0．05)。(3)正常早、中、晚孕及ICP胎盘中均有AE2 mRNA的表达，而以上各胎盘中均未发现BSEP mRNA的表达；晚孕AE2 mRNA表达量高于早、中孕(3．35±2．30 VS 0．81±0．54、1．01±0．89)，差异均有统计学意义(P＜0．05)，ICP组AE2 mRNA表达量高于对照组(5．75±4．37 VS 3．35±2．30)，差异有统计学意义(P＜0．05)。(4)AE2蛋白表达在胎盘组织的滋养细胞。(5)ICP胎盘AE2 mRNA表达量与母血DCA、LCA正相关(r分别为0．476、0．451，P＜0．05)，与脐血DCA、CDCA正相关(r分别为0．523、0．448，P＜0．05)，与TBA和其他胆汁酸水平均无相关性；对照组胎盘组织中AE2 mRNA表达量与母血、脐血中TBA和其他胆汁酸水平均无相关性(r值范围为-0．399～0．523，P＞0．05)。
结论ICP母胎体内总胆汁酸及多数胆汁酸水平均升高。ICP胎盘AE2 mRNA表达增加，由此造成ICP患者胎盘由母体向胎儿方向的胆汁酸转运增强引起胎儿体内胆汁淤积，这可能是ICP胎儿不良结局的原因。
Object To explore the placental expression of AE2、BSEP along with pregnancy and in intrahepatic cholestasis of pregnancy (ICP), as well as their relationships with bile acid levels in maternal and umbilical venous serum. To evaluate the role of placental bile salt transporters in pathological mechanism of ICP.
Methods Total bile acid (TBA) and nine bile acids levels in maternal and umbilical venous serum were measured by velocimetry and HPLC-MS/MS respectively in 20 gravidas complicated with ICP (ICP group) and 20 normal gravidas (control group) of late pregnancy. The placental BSEP and AE2 mRNA expression of 8 early pregnancy, 7 middle pregnancy and two above groups were tested by real time RT-PCR.The locatization of AE2 in placenta was analyzed by immunohistochemistry.
Results (1) Both maternal and umbilical cord serum TBA levels in ICP group were significantly higher than those in control group (46.15±62.82 VS 2.82±1.82μmol/L; 18.23±16.77μmol/L VS 4.79±1.26, P＜0.05). Maternal serum TBA level was significantly higher than that of umbilical serum in ICP group (46.15±62.82 VS 18.23±16.77μmol/L, P＜0.05), and significantly lower than that of umbilical serum in control group (4.79±1.26 VS 2.82±1.82μmol/L, P＜0.05). (2) Matemal serum TCA、CA、TDCA、GCA、GCDCA、TCDCA levels were significantly higher in ICP group than those in control group(P＜0.05), DCA was significantly lower in ICP group than that in control group(P＜0.05); umbilical serum TCA、CA、TDCA、GCA、GCDCA、LCA levels were significantly higher in ICP group than those in control group(P＜0.05); maternal serum TCA、DCA、TDCA、TCDCA、GCA levels were significantly higher than those in umbilical serum of ICP group(P＜0.05);maternal serum DCA、TDCA、LCA levels were significantly higher than those in umbilical cord serum of control group(P＜0.05), TCDCA was significantly lower in maternal serum than that in umbilical serum of control group(P＜0.05). There were significant positive correlations between nine bile acid levels of maternal serum and those of umbilical serum in ICP group(r=0.478～0.952, P＜0.05), as well as positive correlations between GCA、DCA、LCA levels of maternal serum and those of umbilical serum in control group(r=0.585、0.764、0.595, P＜0.05). (3) AE2 mRNA expression was detected both in ICP placenta and in normal placentas along with pregnancy, but we did not find expression of BSEP mRNA in any placenta. AE2 mRNA expression in placenta of late pregnancy was significantly higher than those of early and middle pregnancy (3.35±2.30 VS 0. 81±0.54、1.01±0.89, P＜0.05). AE2 mRNA expression was significantly increased in ICP group than that in control group (5.75±4.37 VS 3.35±2.30, P＜0.05). (4) AE2 expression was detected in trophoblast by immunohistochemistry. (5) There were significant positive correlations between maternal serum DCA、 LCA levels and placental AE2 mRNA expression in ICP group(r=0.476、0.451, P＜0.05), as well as positive correlations between umbilical serum DCA、CDCA levels and placental AE2 mRNA expression (r=0.523、0.448, P＜0.05). There were no significant correlations between maternal or umbilical serum bile acids levels and placental AE2 mRNA expression in control group (r=-0.399～0.523, P＞0.05).
Conclusion Our results suggested that TBA and majority of bile acids increase obviously both in the maternal and fetal profiles in ICP. Increased placental expression of AE2 may improve transporting of bile acids across the placenta toward fetus and result in fetal cholestasis, this is probably involved in the mechanism of poor prognosis of perinatal outcome in ICP.
方法收集妊娠晚期ICP患者(ICP组)及正常晚孕妇女(对照组)胎盘组织及母体静脉血、脐静脉血各20例；收集正常早、中孕胎盘组织各8例、7例。采用速率法检测母、脐静脉血中总胆汁酸(TBA)的含量；采用高效液相色谱质谱联用法检测两组母、脐静脉血中九种胆汁酸：胆酸(CA)，脱氧胆酸(DCA)，鹅脱氧胆酸(CDCA)，牛磺胆酸(TCA)，牛磺脱氧胆酸(TDCA)，牛磺鹅脱氧胆酸(TCDCA)，甘胆酸(GCA)，甘氨鹅脱氧胆酸(GCDCA)，石胆酸(LCA)水平；采用实时荧光定量PCR法测定胎盘组织中BSEP和AE2 mRNA的表达量；采用免疫组织化学法检测AE2在胎盘组织中的细胞学定位。
结果(1)ICP组母血和脐血中TBA水平均高于对照组(46．15±62．82 VS 2．82±1．82μmol／L；18．23±16．77μmol／L VS 4．79±1．26)：ICP组母血中TBA水平高于脐血(46．15±62．82 VS 18．23±16．77μmol／L)，对照组脐血TBA水平高于母血(4．79±1．26 VS 2．82±1．82μmol／L)，以上差异均有统计学意义(P＜0．05)。(2)ICP组母血中TCA、CA、TDCA、GCA、GCDCA、TCDCA水平高于对照组，DCA水平低于对照组；ICP组脐血中TCA、CA、TDCA、GCA、GCDCA、LCA水平高于对照组；ICP组母血中TCA、DCA、TDCA、TCDCA、GCA高于脐血；对照组母血中DCA、TDCA、LCA高于脐血，TCDCA低于脐血；ICP组母血九种胆汁酸成分与脐血相应成分之间均存在正相关(r=0．478～0．952)，对照组母血GCA、DCA、LCA与脐血相应成分之间存在正相关(r分别为0．585、0．764、0．595)，以上差异及相关性均有统计学意义(P＜0．05)。(3)正常早、中、晚孕及ICP胎盘中均有AE2 mRNA的表达，而以上各胎盘中均未发现BSEP mRNA的表达；晚孕AE2 mRNA表达量高于早、中孕(3．35±2．30 VS 0．81±0．54、1．01±0．89)，差异均有统计学意义(P＜0．05)，ICP组AE2 mRNA表达量高于对照组(5．75±4．37 VS 3．35±2．30)，差异有统计学意义(P＜0．05)。(4)AE2蛋白表达在胎盘组织的滋养细胞。(5)ICP胎盘AE2 mRNA表达量与母血DCA、LCA正相关(r分别为0．476、0．451，P＜0．05)，与脐血DCA、CDCA正相关(r分别为0．523、0．448，P＜0．05)，与TBA和其他胆汁酸水平均无相关性；对照组胎盘组织中AE2 mRNA表达量与母血、脐血中TBA和其他胆汁酸水平均无相关性(r值范围为-0．399～0．523，P＞0．05)。
结论ICP母胎体内总胆汁酸及多数胆汁酸水平均升高。ICP胎盘AE2 mRNA表达增加，由此造成ICP患者胎盘由母体向胎儿方向的胆汁酸转运增强引起胎儿体内胆汁淤积，这可能是ICP胎儿不良结局的原因。
Object To explore the placental expression of AE2、BSEP along with pregnancy and in intrahepatic cholestasis of pregnancy (ICP), as well as their relationships with bile acid levels in maternal and umbilical venous serum. To evaluate the role of placental bile salt transporters in pathological mechanism of ICP.
Methods Total bile acid (TBA) and nine bile acids levels in maternal and umbilical venous serum were measured by velocimetry and HPLC-MS/MS respectively in 20 gravidas complicated with ICP (ICP group) and 20 normal gravidas (control group) of late pregnancy. The placental BSEP and AE2 mRNA expression of 8 early pregnancy, 7 middle pregnancy and two above groups were tested by real time RT-PCR.The locatization of AE2 in placenta was analyzed by immunohistochemistry.
Results (1) Both maternal and umbilical cord serum TBA levels in ICP group were significantly higher than those in control group (46.15±62.82 VS 2.82±1.82μmol/L; 18.23±16.77μmol/L VS 4.79±1.26, P＜0.05). Maternal serum TBA level was significantly higher than that of umbilical serum in ICP group (46.15±62.82 VS 18.23±16.77μmol/L, P＜0.05), and significantly lower than that of umbilical serum in control group (4.79±1.26 VS 2.82±1.82μmol/L, P＜0.05). (2) Matemal serum TCA、CA、TDCA、GCA、GCDCA、TCDCA levels were significantly higher in ICP group than those in control group(P＜0.05), DCA was significantly lower in ICP group than that in control group(P＜0.05); umbilical serum TCA、CA、TDCA、GCA、GCDCA、LCA levels were significantly higher in ICP group than those in control group(P＜0.05); maternal serum TCA、DCA、TDCA、TCDCA、GCA levels were significantly higher than those in umbilical serum of ICP group(P＜0.05);maternal serum DCA、TDCA、LCA levels were significantly higher than those in umbilical cord serum of control group(P＜0.05), TCDCA was significantly lower in maternal serum than that in umbilical serum of control group(P＜0.05). There were significant positive correlations between nine bile acid levels of maternal serum and those of umbilical serum in ICP group(r=0.478～0.952, P＜0.05), as well as positive correlations between GCA、DCA、LCA levels of maternal serum and those of umbilical serum in control group(r=0.585、0.764、0.595, P＜0.05). (3) AE2 mRNA expression was detected both in ICP placenta and in normal placentas along with pregnancy, but we did not find expression of BSEP mRNA in any placenta. AE2 mRNA expression in placenta of late pregnancy was significantly higher than those of early and middle pregnancy (3.35±2.30 VS 0. 81±0.54、1.01±0.89, P＜0.05). AE2 mRNA expression was significantly increased in ICP group than that in control group (5.75±4.37 VS 3.35±2.30, P＜0.05). (4) AE2 expression was detected in trophoblast by immunohistochemistry. (5) There were significant positive correlations between maternal serum DCA、 LCA levels and placental AE2 mRNA expression in ICP group(r=0.476、0.451, P＜0.05), as well as positive correlations between umbilical serum DCA、CDCA levels and placental AE2 mRNA expression (r=0.523、0.448, P＜0.05). There were no significant correlations between maternal or umbilical serum bile acids levels and placental AE2 mRNA expression in control group (r=-0.399～0.523, P＞0.05).
Conclusion Our results suggested that TBA and majority of bile acids increase obviously both in the maternal and fetal profiles in ICP. Increased placental expression of AE2 may improve transporting of bile acids across the placenta toward fetus and result in fetal cholestasis, this is probably involved in the mechanism of poor prognosis of perinatal outcome in ICP.
引文
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24．张力，刘淑芸，时青云，等．雌激素受体α基因多态性与妊娠肝内胆汁淤积症相关性研究[J]．中华妇产科杂志，2006；41(5)：307-310．
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26．陈富强，范丽安，许玲娣，等．人类白细胞抗原-DRBI基因多态性与妊娠期肝内胆汁淤积症的相关性研究[J]．中华妇产科杂志，2002；37：519-522．
27．张力，刘淑芸，陈强，等．成都地区妊娠肝内胆汁淤积症家庭HLA-DPA1基因多态性研究[J]．四川大学学报(医学版)，2003；34(3)：530-532．
28．彭冰，陈强，张力，等．成都地区妊娠期肝内胆汁淤积症与人类白细胞抗原一DQA1的相关性研究[J]．中华医学遗传学杂志，2006；23(5)：555—557．
29．张力，刘淑芸，陈强，等．CYP17和CYP3A4基因多态性与成都地区妊娠期肝内胆汁淤积症关系的研究[J]．四川大学学报(医学版)，2006；37(4)：551—553．
30．张力，刘淑芸，刘兴会，等．雌激素代谢基因CYP1A2和COMT单核苷酸多态性与妊娠期肝内胆汁淤积症关系的研究[J]．现代妇产科进展，2006；15(6)：430-433．
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18. Jansen PL, Sturm E. Genetic cholestasis, causes and consequences for hepatobiliary transport[J]. Liver International, 2003;23(5):315-22.
19. Klomp LW, Vargas JC, van Mil SW, et al. Characterization of Mutations in ATP8B1 Associated With Hereditary Cholestasis [J]. Hepatology, 2004;40(1):27-38.
20. Jirsa M, Cebecauerova D, Budisova L, et al. Indel in the FIC1 /ATP8B1 gene—a novel rare type of mutation associated with benign recurrent intrahepatic cholestasis[J]. Hepatology Research, 2004;30:l-3.
21. Bull LN, van Eijk MJ, Pawlikowska L, et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis [J]. Nature Genetics. 1998; 18(3):219-224.
22. Mullenbach R, Bennett A, Tetlow N, et al. ATP8B1 mutations in British cases with intrahepatic cholestasis of pregnancy [J]. Gut, 2005;54 (6):829-34.
23. Eloranta ML, Heiskanen JT, Hiltunen MJ, et al. Multidrug resistance 3 gene mutation 1712delT and estrogen receptor alpha gene polymorphisms in Finnish women with obstetric cholestasis [J]. European Journal of Obstetrics, Gynecology, & Reproductive Biology, 2002; 105(2): 132-5.
24．张力，刘淑芸，时青云，等．雌激素受体α基因多态性与妊娠肝内胆汁淤积症相关性研究[J]．中华妇产科杂志，2006；41(5)：307-310．
25. Mella JG, RoschmannE, Glasinovic JC, et al. Exploringthe genetic roleofthe HLA—DPBI locus in Chileans with intrahepatic cholestasis of pregnancy[J]. J Hepatol, 1996;24: 320-323.
26．陈富强，范丽安，许玲娣，等．人类白细胞抗原-DRBI基因多态性与妊娠期肝内胆汁淤积症的相关性研究[J]．中华妇产科杂志，2002；37：519-522．
27．张力，刘淑芸，陈强，等．成都地区妊娠肝内胆汁淤积症家庭HLA-DPA1基因多态性研究[J]．四川大学学报(医学版)，2003；34(3)：530-532．
28．彭冰，陈强，张力，等．成都地区妊娠期肝内胆汁淤积症与人类白细胞抗原一DQA1的相关性研究[J]．中华医学遗传学杂志，2006；23(5)：555—557．
29．张力，刘淑芸，陈强，等．CYP17和CYP3A4基因多态性与成都地区妊娠期肝内胆汁淤积症关系的研究[J]．四川大学学报(医学版)，2006；37(4)：551—553．
30．张力，刘淑芸，刘兴会，等．雌激素代谢基因CYP1A2和COMT单核苷酸多态性与妊娠期肝内胆汁淤积症关系的研究[J]．现代妇产科进展，2006；15(6)：430-433．
31. Eloranta ML, Heiskanen J, Hiltunen M, et al. Apolipoprotein E alleles in women with intrahepatic cholestasis of pregnancy[J]. Scandinavian Journal of Gastroenterology, 2000;35(9):966-8.