先天性巨结肠症免疫缺陷相关基因的精细定位及其功能研究

Hirschsprung disease（HSCR）is a common disease in children caused by the developmental disorder of the enteric nervous system with high incidence rates. At present, the major treatment for HSCR is to remove the abnormal section of the colon. However, a part of HSCR patients may develop enterocolitis despite they had successful surgeries or not, a condition referred to as Hirschsprung-associated enterocolitis (HAEC) which will lead to die in some patients and is a dominant cause of death for HSCR patients. To date, the pathogenesis of HAEC is still not very clear with a lot of controversy, of which immunodeficiency is one of the important causes. Our previous research found that AGH rats used as animal model of HSCR showed a small spleen phenotype with decreased white blood cell counts but not in LEH rats with the different genetic backgrounds, which suggested that genetic factors may be an important cause of HAEC, which result in the immunity defect by affecting spleen development. In the previous research, a significant QTL that affected the spleen development has been identified by QTL analysis in F2 population produced by breeding the AGH and LEH strains..In this project, we try to identify the specific genes responsible for the immunity defect by increased F2 population, producing sub-congenic strains and RNA-Seq technique. Next we will validate the function of specific gene by producing the knockout mouse, then which will be used as animal model to analysis the molecular mechanism that the responsible gene affected the spleen by tracing the dynamic development of spleen cells. This project will likely put forward a novel explanation that HAEC happens and help to promote the prevention and care of HAEC.

先天性巨结肠症是小儿常见的肠神经系统发育障碍疾病，主要通过手术切除无神经节肠段治疗。但无论手术与否，总有部分病例并发巨结肠小肠结肠炎（HAEC），严重者导致死亡，是巨结肠症患者死亡的主要原因。迄今为止，关于HAEC的发病机理仍存在争议，其中免疫缺陷是影响其发病的原因之一。前期研究发现携带Ednrb基因突变的AGH大鼠呈现小脾脏等免疫缺陷表型，但在LEH鼠系表型正常，提示由遗传因素影响脾脏发育、导致免疫缺陷可能是HAEC发生的一个原因。前期研究已鉴定一个达显著水准的QTL，本研究将通过增加F2代数量、标记辅助制作亚导入系，结合RNA-Seq技术精细定位主效基因，用基因敲除小鼠进行候选基因功能验证，并以此小鼠及2种大鼠为模型追踪脾脏发育期细胞增殖、凋亡情况及相关基因表达丰度，确定候选基因影响脾脏发育的内在机制。本研究将为阐明HAEC的发生机理提供一定的理论依据，有助于对其预防和治疗。

先天性巨结肠(Hirschsprung's disease, HSCR)又称先天性肠无神经节细胞症, 是导致儿童便秘及低位不完全肠梗阻的常见外科疾病, 发病率约为1/5 000，HSCR发病率呈逐年上升趋势, 严重危害新生儿健康。但是该病致病因素复杂，是一种典型的多基因疾病，目前尚未完全研究透彻。在前期研究中我们发现，EDNRB基因突变不仅会出现巨结肠表型，其脾脏大小也会有所改变。用AGH-Ednrbsl公鼠与LEH-Ednrbsl 母鼠杂交得到F1代，再互交产生F2代，以脾脏比重作为表型数据进行QTL分析鉴定影响脾脏发育及免疫缺陷相关基因，在10号染色体鉴定了1个达到显著水准的QTL并确定了候选基因。另外，利用EDNRB基因突变小鼠进行脾脏转录组测序和肠道单细胞测序，尝试找到脾脏表型改变的内在机制，并进一步利用最新的单细胞测序技术去挖掘先天性巨结肠可能的致病基因。在脾脏转录组研究中，共筛选出了121个差异表达的基因，共85个上调基因，36个下调基因，对差异基因集进行GO功能富集分析和KEGG通路富集分析，发现在Ednrbmlzcm小鼠中与凋亡相关的p53信号通路（p53 signaling pathway）、凋亡通路（Apoptosis - multiple species）等的相关基因表达量升高，Hippo 信号通路（Hippo signaling pathway）相关基因上调，此通路抑制细胞生长，对器官大小和体积的调控有影响，因此可能与脾脏组织变小有关。在单细胞测序研究中，我们绘制了突变型和野生型小鼠结肠的单细胞图谱，鉴定了神经嵴细胞来源的神经元细胞和神经胶质细胞。同时，对这两种细胞的差异表达情况进行了分析，获得了单细胞水平的差异表达基因（如神经元细胞：Iqsec3、Snhg11、Vip、Chrna7、Ralyl等；神经胶质细胞：Gjc3、Cdh19、Sox10、Cmtm5、Megf10等）。本研究利用遗传筛选、转录组测序、单细胞解析三种手段，发现了大量可能导致脾脏表型改变和巨结肠发生的基因和通路，这将为今后巨结肠及相关免疫缺陷致病基因的发掘提供重要的依据。