基于类器官模型探究胆汁淤积症新致病基因MYO5B影响小鼠肝细胞极性的机制

Disruption of hepatocyte polarity is one mechanism of hereditary cholestasis, the vital cause of pediatric liver diseases. Recently, we identified MYO5B as a new pathogenic gene of hereditary cholestasis. Patients with MYO5B defect showed abnormal location and function of the major canalicular transporters, suggesting hampered hepatocyte polarity. However, MYO5B function in the liver was unclear, let alone lacking ideal models to study hepatocyte polarity. Organoids from liver tissues can differentiate into solid, functional hepatocytes via three-dimensional cultivation, indicating a new and effective method to study hepatocyte polarity and related genetic defect. In this study, firstly we plan to construct liver organoids from Myo5b liver specific knockout mice to recapitulate the observations in MYO5B deficient cholestasis; and then by histochemistry, transcriptome sequencing, quantitive proteomics and bile secreting experiments etc, we try to explore how MYO5B affects organoid cell polarity formation and related pathway changes; next, we plan to transfect the defect organoids with Myo5b Lentivirus to access the potential of gene correction. This study tries to elucidate the mechanisms of hampered polarity and canalicular transporter abnormalities, and findings of which will provide new ideas for therapies of MYO5B defect and other liver diseases.

遗传性胆汁淤积症是儿童肝病的重要病因。申请者新近鉴定MYO5B可致遗传性胆汁淤积症谱系，并发现肝细胞毛细胆管面主要转运体定位及功能异常，推测肝细胞极性受损所致。MYO5B在肝细胞内具体作用尚不清楚，且缺乏研究肝细胞极性的理想模型。肝组织类器官培养可在体外形成保持极性的成熟肝细胞，可望用于肝细胞极性及相关疾病研究。本研究拟以同窝野生型小鼠来源的肝类器官为对照，构建Myo5b肝特异性敲除小鼠相应肝类器官，通过组织化学、免疫印迹、胆汁酸分泌实验等观察类器官分化后肝细胞极性受损情况，并应用Myo5b全序列重组慢病毒感染缺陷肝类器官进行体内外分化实验，观察肝细胞极性恢复情况；进一步通过转录组测序、定量蛋白组学等手段研究MYO5B缺陷在肝类器官细胞极性形成过程中分子通路改变；从而阐明MYO5B缺陷导致肝细胞极性受损、毛细胆管面主要转运体异常的机制，为MYO5B缺陷及其它肝病的治疗提供思路。

MYO5B缺陷是我们新近发现导致遗传性胆汁淤积症的重要原因之一，患者肝细胞极性面重要转运体胆盐外运泵(BSEP)继发定位异常、但具体机制并不清楚，当时亦无与该表型对应的体外模型。本研究原计划前期构建的Myo5b肝脏特异性敲除小鼠(Myo5b-cko)，构建肝类器官以期复现胆汁淤积发生过程并研究相关致病机制。然而，Myo5b-cko衍生肝类器官相比野生型同窝对照，相同时间点的形态、增殖速度、增殖及肝祖细胞标记基因的表达并无差异。分化后肝类器官细胞均无法稳定表达Bsep，但与之发育时间点相近的1周龄及成年小鼠肝组织荧光染色显示，Myo5b-cko敲除并不影响Bsep定位及表达丰度、细胞极性不受影响。进一步对不同传代次数肝类器官及不同时间点小鼠肝组织mRNA分析发现，相同条件下Myo5b-cko相比对照组，肝细胞总体基因表达PCA并无差异、差异表达基因/通路极少，细胞极性相关基因 (Bsep, Ntcp, Zo-1)及相关通路 (Apical surface)不受影响，与同期进行的Myo5b-cko小鼠大体表型实验结果一致；但向Myo5b-cko及对照小鼠注射含MYO5B肝病患者高频变异（R824C）的人MYO5B变异序列腺病毒，可观察到肝细胞极性标志Bsep部分分布异常、与患者表型类似。因此认为，Myo5b敲除不是MYO5B缺陷肝病的致病机制，相关动物/类器官模型无法模拟胆汁淤积表型。参考腺病毒注射结果，我们进而构建病例相应的Myo5b点突变小鼠模型Myo5bR825C/R825C，目前在进行种群扩繁中。初步实验发现，Myo5bR825C/R825C暂未观察到异常，但给与1%胆酸进行胆汁淤积负荷后，该小鼠相较野生型对照体重下降更多。本研究发现MYO5B敲除不影响肝细胞极性、与原先假设不符，但我们据此设计了的更接近肝病患者的点突变小鼠，为进一步机制研究、疾病新药筛选提供思路。

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