基于miRNA表达异常导致Th1/Th2免疫失调的PBC发病机制及中医补虚化瘀治法研究

Based on the "liver" "liver storing blood" theory and clinical practice, proposed and confirmed Buxu Huayu method has good effect in treating patients with PBC, these previous studies also confirmed the Th1/Th2 immune dysfunction is an important factor in the pathogenesis of PBC. This study will use the miRNA microarray technology, testing for PBC patients, organization model animal and whole blood samples. Screening and the pathogenesis of PBC associated and alternative miRNAs has regulatory effect on Th1/Th2 cells, then the candidate expression of miRNA further verification and detection of Th1/Th2 specific transcription factor T-bet/GATA-3 gene, protein by dnTGF-βRII transgenic mouse model. To clarify the abnormal expression of miRNA leading to Th1/Th2 immune dysregulation contributes to the pathogenesis of PBC. Then the model of Buxu Huayu Prescription and tonic, removing blood stasis and role of in-depth study, effect of treatment on biochemical, pathological, Th1/Th2 cytokines and alternative miRNA, T-bet/GATA-3 gene, protein expression, Clearly the internal molecular mechanism of Chinese medicine of Buxu Huayu method, looking for therapeutic target in PBC Buxu Huayu method.

我们基于“肝主疏泄”“肝藏血”理论和临床实践，提出并证实补虚化瘀法对PBC患者具有良好的辨治效果，既往实验研究也证实了Th1/Th2免疫失调是PBC发病的重要因素。本次研究将采用miRNA微阵列芯片技术，对PBC患者、模型动物组织及全血样本进行检测，筛选出与PBC发病关联且对Th1/Th2细胞具有调控作用的备选miRNAs，其次通过dnTGF-βRⅡ转基因模型小鼠对备选miRNA 基因表达进一步验证并检测Th1/Th2特异性转录因子T-bet/GATA-3基因、蛋白表达，阐述miRNA表达异常导致Th1/Th2免疫失调导致PBC的发病机制。继而基于此模型对补虚化瘀方以及补虚、化瘀拆方作用深入探讨，研究相应治法对生化指标、病理、Th1/Th2细胞因子以及备选miRNA、T-bet/GATA-3基因、蛋白表达的影响，以阐明中医补虚化瘀法的内在分子效应机制，寻找补虚化瘀法治疗PBC的作用靶点。

我们基于“肝主疏泄”“肝藏血”理论和临床实践，提出并证实补虚化瘀法对PBC患者具有良好的辨治效果，既往实验研究也证实了Th1/Th2免疫失调是PBC发病的重要因素。本次研究采用miRNA微阵列芯片技术，对PBC患者外周血样本进行检测，筛选出7个与PBC发病相关联并且和Th1/Th2细胞、胆管上皮细胞有调控作用的候选miRNA，分别为hsa-miR-21-3p、hsa-miR-148-3p、hsa-miR-223-3p、hsa-miR-125a-5p、hsa-miR-193a-3p、hsa-miR-378a-3p和hsa-miR-150-5p。进一步扩大临床样本显示，和正常人相比，hsa-miR-21-3p、hsa-miR181a-3p表达在PBC中均上调，而hsa-miR-148-3p表达下调，并且hsa-miR148a-3p表达与IgM指标呈负相关。在体外细胞实验中，进一步探讨中药补虚化瘀法治疗PBC的作用机制研究发现，LPS诱导人胆管上皮细胞炎症反应模型，主要以分泌CXCL10 趋化因子和IL-8细胞因子为主，该模型存在miRNA21-3p表达下降，而补虚化瘀治法中药整方及补虚、化瘀拆方可以显著下调LPS引起的CXCL10 mRNA基因和IL-8蛋白分子的表达的升高，作用机制可能与调控miRNA21-3p的表达有关。该研究阐述了中医补虚化瘀法的内在分子效应机制，寻找出了补虚化瘀法治疗PBC的作用靶点，为更深入探索中药复方的作用机制提供了依据。