血清胆汁酸谱检测及其在妊娠肝内胆汁淤积症中的作用

Intrahepatic cholestasis of pregnancy (ICP) is a complication of pregnancy that would cause serious damages to mother and infant, especially to perinatal infant. Its pathogenesis has not been clearly understood, neither is the specific diagnostic indicator and grading criteria. Researches showed that abnormal bile acid metabolism occured in hepatobiliary diseases. Furthermore, different diseases had their characterized bile acids profiling. Because of some bile acids have very low concentration in vivo, and bile acids themselves have no electrochemical signals. But with the coexisting of nicotinamide adenine dinucleotide (NAD+), bile acids are converted to 3-ketosteroids under the catalysis of 3α-hydroxysteriod dehydrogenase (3α-HSD) with concomitant reduction of NAD+ to reduced nicotinamide adenine dinucleotide (NADH). With an appropriate voltage applied on the working electrode, NADH is oxidized to form NAD+, which results in electrochemical signal and could analyze the serum bile acids profiling with high sensitivity by the electrochemical detection. Polydimethylsiloxane (PDMS) chips have the characteristics of low cost and fast separation. This project plans to interconnect the high efficiency of PDMS chip in separation and high sensitivity of electrochemical method in detection to develop a new method for the analysis of bile acids profiling. And next, the method could be applied to the detection of serum bile acids profiling in ICP patients. The aim is to develop the specific diagnosis and grading criteria of ICP and select the specific biomarkers for ICP, which could also provide a prospective basis to understand the pathogenesis of ICP.

妊娠肝内胆汁淤积症（ICP）是一种对母婴，特别是围生儿造成严重危害的妊娠并发症。其发病机制不明，也无特异的诊断与分度指标。研究显示，肝胆疾病时胆汁酸代谢异常，且不同疾病有其特征的胆汁酸谱。由于某些胆汁酸在体内含量极低，且胆汁酸本身又无电化学信号，但在氧化型辅酶I (NAD+)共存下，能被3α-羟类固醇脱氢酶(3α-HSD )催化脱氢生成3-酮类固醇，NAD+还原为还原型辅酶I (NADH)。在工作电极上施加恒电位使NADH被氧化为NAD+，产生电化学响应，从而可通过电化学法实现血清胆汁酸谱的高灵敏度检测。PDMS芯片具有成本低、快速分离等特点。本项目将高效分离的PDMS芯片和高灵敏度的电化学检测偶联建立胆汁酸谱检测新方法，用于ICP患者血清胆汁酸谱分析，以期建立ICP特异诊断和分度模式，有望筛选出特异的ICP生物标志物，并为理解ICP的发病机制提供依据。

妊娠期肝内胆汁淤积症（intrahepatic cholestasis of pregnancy，ICP）是一种对围生儿造成严重危害的妊娠期并发症，目前其发病机制不明且缺乏特异的诊断和鉴别诊断指标。本研究以ICP 的准确诊断和鉴别诊断为目标，结合电化学检测方法、PDMS 芯片分离技术以及超高效液相色谱-三重四级杆飞行时间质谱技术（UPLC-TripleTOF -MS/MS），建立了简便快速检测血清胆汁酸的新方法，并基于此寻找用于诊断和鉴别诊断ICP的灵敏特异的胆汁酸生物标志物。该研究首先建立了快速检测血清总胆汁酸的电化学检测方法，并成功应用于临床真实血清样本中的胆汁酸检测。结果显示，该方法与目前临床常用的检测血清总胆汁酸的酶循环法具有很好的相关性。在此基础上，本研究还建立了PDMS 芯片结合电化学法检测血清胆汁酸的方法，该方法也被成功用于临床真实样本血清胆汁酸检测。研究还采用超高效液相色谱-飞行时间质谱技术对ICP 患者和妊娠期无症状高胆汁酸血症(asymptomatic hypercholanaemia of pregnancy，AHP)孕妇血清中包含55种分类胆汁酸的胆汁酸谱进行了全面分析，发现ICP 患者与正常孕妇和AHP孕妇的血清胆汁酸谱均差异显著。我们对数据进行偏最小二乘法-判别分析，筛选出GCA，Gtri-3和Ttri-2三种用于ICP 诊断和DCA、α-MCA、Gtri-1和di-S-3四种用于ICP与AHP鉴别诊断的灵敏特异的胆汁酸标志物。Gtri-3和Ttri-2联合诊断标志物对ICP进行诊断的灵敏度为92.9%，特异性为90.5%。ICP和AHP鉴别诊断的灵敏度达到97.8%，特异度为94.3%。本研究有助于临床对ICP的准确诊断与鉴别诊断，为临床选择恰当治疗方案提供实验室依据，对改善母婴预后，提高人口质量具有重要意义。本项目培养博士研究生1名，硕士研究生3名；发表SCI论文5篇，还有一篇SCI论文即将投稿。申请国家发明专利两项，其中一项已授权。