胆道炎症诱导胆管细胞miRNA网络失衡参与肝内胆管结石形成特征分析及大黄灵仙胶囊的调节效应

Biliary inflammation is a primary intrahepatic bile duct stones basic pathological process, bile duct cells directly involved. Surgery is the primary means of treatment of this disease,but the recurrence of stones is a problem never unsolved,even though surgical technology continues to progress. Recent studies have found that the single-stranded miRNA is a small molecule RNA 21-25 nt, which is involved in complex gene regulation and plays an important role in disease progression. Thus, the group proposed bile duct inflammation induced cell miRNA network imbalance, through complex regulatory mechanisms involved in the formation of stones in the bile duct.More over our previous studies have found rhubarb Ling Xian capsules can reduce the recurrence rate of postoperative stone stable CYP7A1 mRNA expression, prevent stone formation. Therefore, the project built from rat bile duct infection status LPS injection, using a variety of research tools, specifically bile duct inflammation characterized by cellular miRNA network, while revealing rhubarb Ling Xian Capsule control target miRNA, and to clarify its expression by regulating miRNA and play effector mechanisms, interpretations regulating effect of Dahuang Ling Xian capsule cholangiocarcinoma miRNA networks for its treatment of the disease provide a deeper scientific basis, to complement Western medicine did not catch.

胆道炎症是原发性肝内胆管结石的基本病理过程，胆管细胞直接受累，外科手术是本病当前主要治疗手段，但术后结石复发一直是未能解决的难题，尽管手术技术不断进步。新近研究发现的miRNA是21-25 nt的单链小分子RNA，其参与复杂的基因调控，在疾病进展中起重要作用。由此，课题组提出胆道炎症诱导胆管细胞miRNA网络失衡，后者通过复杂的调控机制，参与胆管内结石形成，且课题组前期研究发现大黄灵仙胶囊能降低患者术后肝内结石复发率，稳定CYP7A1 mRNA 表达，预防结石形成。因此，本项目从大鼠胆总管注射LPS构建胆管感染状态，采用多种研究手段，明确胆道炎症诱导胆管细胞miRNA网络失衡特征，揭示大黄灵仙胶囊调控的靶miRNA，并阐明其通过调节miRNA表达而发挥的效应机制，诠释大黄灵仙胶囊对胆管细胞miRNA网络的调节效应，为其治疗本病提供更深刻的科学依据，以补西医之未逮。

原发性肝胆管结石主要治疗手段为外科手术，但难以避免术中对胆管及肝脏的损伤、术后结石的残留与复发等问题。本项目从中医药宝库中挖掘出有效中药方剂，形成大黄灵仙胶囊，经过前期临床验证，证实大黄灵仙胶囊对降低原发性肝胆管结石患者术后结石复发率有确切疗效。因此，本项目为进一步探明结石成因及大黄灵仙胶囊通过调节靶 miRNA表达而发挥的效应机制，构建了兔胆石症动物模型确定大黄灵仙胶囊的整体疗效；分离并培养了大鼠胆管细胞；进一步构建了胆管细胞炎症模型，完成miRNA芯片分析及生物信息学分析，构建大黄灵仙胶囊通过影响miRNA发挥的综合信息网络；并挑选了关键miRNA在细胞及动物层面进行表达量验证；开展了大鼠体内miRNA干扰实验。研究中确定了胆管细胞炎症应激参与胆道病理性重塑，明确了大黄灵仙胶囊防治胆石症的整体疗效，确定了大黄灵仙胶囊防治胆石症的miRNA生物信息网络，确定了大黄灵仙胶囊可能调控的miRNAs，证实大黄灵仙胶囊在胆管细胞信号转导、有机化合物反应及缺氧应激等生物学过程中，可调节细胞质、内质网及膜筏等细胞组份，调控信号转导活性、转录调控区的 DNA 结合及电压门控钾通道活性等发挥分子生物功能，明确了胆道炎症诱导胆管细胞 miRNA 网络失衡参与胆管结石成石特征。阐明大黄灵仙胶囊通过调节胆管细胞靶 miRNA表达而发挥的效应机制，通过构建miR-152-3p腺病毒干扰质粒，对miR-152-3p在胆道炎症进程中调控胆道上皮细胞上皮-充间质转化的作用进行了进一步分析，为后续研究大黄灵仙胶囊作用miRNA 精细调控肝内胆管结石形成的内涵夯实基础。