RET/CXCR4轴介导神经嵴衍生细胞迁移及其在先天性肠无神经节细胞症发病中的作用

Hirschsprung's disease (HSCR) is a severe congenital malformation of the gastrointestinal tract, which dues to the abnormal development of neural crest. Recent studies have showed that RET gene mutation is most closely related to the development of HSCR, but the mechanism is unclear. Our preliminary studies have found that the expression of CXCR4 is regulated by RET in neuroblastoma cell lines which is derived from neural crest cells. In addition, CXCR4 plays a key role in the invasion and metastasis of neuroblastoma. Further research found that the expression of RET and CXCR4 in the aganglionic colon tissue of HSCR patients were significantly decreased compared with that in normal colon tissue. On this basis, we put forward the hypothesis that RET gene could regulate CXCR4 expression thus promoting the migration of the precursor cells of enteric nervous system derived from neural crest cells, and which plays an important role in the development of the enteric nervous system; furthermore, the abnormality of RET/CXCR4 axis reduces the migration of the precursor cells of enteric nervous system thus giving rise to the development of HSCR. To examine the key molecular mechanism leading to the development of HSCR, we plan to use the neural crest stem cells and gene knockout mice model to explore whether the mutations of RET could affect the migration of neural crest-derived cells by suppression of the CXCR4 expression, or by any other potential molecular pathways to affect the development of the enteric nervous system thus leading to the development of HSCR.

先天性肠无神经节细胞症（HSCR）是胚胎期神经嵴发育异常导致的严重消化道先天畸形，RET基因异常与HSCR的发病密切相关，但其具体分子机制尚不明确。课题组前期研究发现，RET基因能调节神经嵴来源的神经母细胞瘤CXCR4的表达而介导神经母细胞瘤的侵袭转移。进一步研究发现，HSCR患者病变结肠组织RET、CXCR4表达水平均显著低于正常结肠。在此基础上我们提出假设：RET基因可能通过调节CXCR4表达介导神经嵴衍生的肠神经系统前体细胞在肠道迁移而参与肠神经系统发育，RET/CXCR4轴异常导致肠神经系统前体细胞迁移减少进而导致HSCR的发生。本项目拟通过临床分子检测、体外细胞实验、基因敲除小鼠实验等研究手段，明确RET基因异常是否通过调控CXCR4表达而影响神经嵴衍生细胞的迁移，或通过其他潜在分子途径影响肠神经系统发育而导致HSCR的发生，初步探讨RET基因异常导致HSCR发病的关键分子机制。

先天性肠无神经节细胞症（HSCR）是胚胎期神经嵴发育异常导致的严重消化道先天畸形，RET基因异常导致神经嵴干细胞在肠道迁移障碍与HSCR的发病密切相关，但其具体分子机制尚不明确。前期研究发现，RET基因能调节神经嵴来源的神经母细胞瘤CXCR4的表达而介导神经母细胞瘤的侵袭转移，且HSCR患者病变结肠组织RET、CXCR4表达水平均显著低于正常结肠，而趋化因子受体CXCR4在胚胎发育细胞迁移中发挥重要作用。在此基础上我们提出假设：RET基因可能通过调节CXCR4表达介导神经嵴衍生的肠神经系统前体细胞在肠道迁移而参与肠神经系统发育，RET/CXCR4轴异常导致肠神经系统前体细胞迁移减少进而导致HSCR的发生。本项目通过临床分子病理检测证实HSCR患者结肠组织痉挛段RET、CXCR4的表达水平较正常段显著降低，且两种蛋白共同表达于神经节细胞，线性回归分析提示两者的表达呈一定程度的相关性。进一步采用原代培养的小鼠肠神经嵴干细胞作为研究对象，通过下调神经嵴衍生细胞（包括肠神经嵴干细胞、神经嵴衍生细胞）Ret基因的表达，证实Ret基因可通过其下游转录因子Etv4/5调节趋化因子受体Cxcr4表达，抑制Ret/Cxcr4轴或者直接使用拮抗剂抑制Cxcr4受体均能导致神经嵴衍生细胞的迁移障碍。在此基础上，通过构建Ret基因敲除小鼠HSCR疾病模型研究发现：Ret基因敲除小鼠通过抑制Ret/Cxcr4轴影响神经嵴干细胞及肠神经系统前体细胞在肠道中的迁移，进而导致肠神经系统的发育障碍而出现先天性肠无神经节细胞症。本项目首次证实Ret基因在是通过其下游转录因子Etv4/5调节趋化因子受体Cxcr4表达从而介导神经嵴干细胞及肠神经系统前体细胞的迁移。阐明了Ret基因异常导致先天性肠无神经节细胞症发生的关键分子机制。为HSCR疾病的临床病理诊断、指导病变结肠手术切除范围，及产前诊断提供新的策略思路。