TCDC/CDC作为胆道闭锁早期诊断的血清标记物及其影响肝损伤的机制研究

Biliary Atresia (BA) is a severe neonatal cholestastic disease, with unknown etiology and no effective diagnostic markers. Early diagnosis and Kasai operation greatly improve the outcome of BA. Our research has discovered that bile acids content in dried blood spots of BA infants were significantly elevated right after birth. Further investigation of sera from BA before Kasai operation (about 2 month of age) indicated that the ratio of taurochenodeoxycholate/chenodeoxycholate (TCDC/CDC) increased significantly in BA infants compared to age and gender matched neonatal hepatitis syndrome (NH) infants. The aim of this study is to identify whether serum TCDC/CDC could be used as an early diagnosis marker for BA (before 1 month of age) and the impact of different bile acids profiling on liver damage. Following research will be conducted: ① Sera of BA and NH before 1 month of age will be collected and MRM will be introduced to profile serum bile acids. ② RT-PCR and MRM will be used to detect the gene expression and protein content of hepatic bile acids synthesis, transport, and nuclear receptor genes. ③ The impact of different bile acids profiling and gene expression on liver damage will be investigated using RRV induced BA model. This research will help us to look for early diagnosis markers of BA, figure out the molecular mechanism of the different bile acids spectrum and its impact of liver damage, provide new clues to the treatment of BA and other neonatal cholestasis.

胆道闭锁（BA）是一种严重的新生儿胆汁淤积疾病，病因未明且缺乏有效的诊断标记物。早期诊断和及早手术能改善其预后。前期研究发现BA患儿刚出生即存在胆汁酸含量显著升高;且出生2月左右其血清牛磺脱氧鹅胆酸/脱氧鹅胆酸（TCDC/CDC）的比值显著高于婴儿肝炎综合征（婴肝）。为进一步研究血清TCDC/CDC能否作为BA早期诊断分子标记物及胆汁酸谱差异对肝损伤的影响，拟进行如下研究：①收集出生在1月以内的胆汁淤积婴儿血清(含BA和婴肝)，MRM检测TCDC/CDC作为BA早期诊断分子标记物的有效性；②RT-PCR和MRM检测肝脏胆汁酸合成、转运和核受体基因的表达和蛋白含量，寻找导致胆汁酸谱差异的靶基因；③利用RRV诱导的BA小鼠模型研究胆汁酸谱和靶基因干预对肝损伤的影响。本研究有助于寻找BA早期诊断的标记物、揭示差异胆汁酸谱的分子机制及其影响肝损伤的机制，为BA和其它胆汁淤积疾病的治疗提供新线索。

背景：胆道闭锁（BA）早期诊断是临床关注的热点和难点。因其与其他婴儿胆汁淤积疾病，特别是婴儿肝炎综合征（NHS），具有相似的临床和生化特征，目前尚无有效的早期诊断方法。由于BA和NHS导致胆汁淤积的机制不同，我们推测其胆汁酸谱可能存在差异，并且导致BA和NHS的肝损伤程度不同。.方法：应用超高效液相色谱-串联质谱技术（UPLC-MS/MS）检测肝脏和血浆中胆汁酸谱；RT-PCR和多反应检测技术（MRM）定量检测肝脏胆汁酸代谢、转运和核受体的基因和蛋白表达；最后利用原代人肝细胞和胆管结扎的小鼠模型研究胆汁酸硫酸化酶SULT2A1对肝脏的保护作用。.结果：通过本课题研究，建立了同时定量检测40种胆汁酸的质谱方法。对BA、NHS和正常对照的血浆和肝脏胆汁酸谱检测，我们在BA和NHS中定量到19种不同的胆汁酸，其中初级结合胆汁酸（GCA、TCA、GCDCA、TCDCA）是其中的主要胆汁酸。多维统计分析发现BA和NHS血浆胆汁酸谱存在显著差异，特别是TCDCA/CDCA的比值，在BA中显著高于NHS和正常对照，TCDCA/CDCA区分BA和NHS的ROC曲线下面积达0.92，并且对出生早期的患儿（出生20-40天）诊断效果更为显著，达到0.96。肝脏胆汁酸谱相关基因表达检测结果显示BA和NHS的胆汁酸转运蛋白（BSEP和MDR3）和核受体（FXR和SHP）表达存在显著差异，胆汁酸合成限速酶CYP7A1表达并无显著差异。进一步对肝脏胆汁酸代谢酶的MRM检测发现胆汁酸硫酸化酶SULT2A1表达在肝纤维轻的患儿中高于肝纤维化重的患儿，尿液硫酸化胆汁酸GCDCA-3S检测显示其含量随着纤维化增加而减少。非硫酸化胆汁酸GCDCA能导致原代人肝细胞凋亡，而硫酸化胆汁酸GCDCA-3S并不引起肝细胞凋亡。然而我们用SULT2A1慢病毒表达载体干预胆管结扎小鼠并没有发现肝纤维化的显著差异。.结论：血浆胆汁酸谱在BA和NHS中存在显著差异，血浆TCDCA/CDCA比值可以作为BA早期诊断标记物；BA和NHS血浆胆汁酸谱的差异可能是由于两者胆汁酸转运蛋白表达差异引起的；SULT2A1能极大降低胆汁酸的肝细胞毒性，可能作为BA患儿Kasai术后的干预靶标。