特定转录因子直接诱导成纤维细胞成为痛相关感觉神经元的研究

Direct conversion of somatic cells to specific neural cells by defined factors is an emerging field in developmental neurobiology, which is clinically useful to generate specific neurological disease relevant cell types from distinct patients (such as fibroblasts) for human disease modeling and therapy. The perception of pain is initiated by distinct nociceptive/pain related sensory neurons (nociceptors) which are comprised of a variety of sensory ion channels and receptors. All these neurons are initially marked by the expression of the nerve growth factor receptor TrkA, and located in the dorsal root ganglia (DRG) and the trigeminal ganglia (TG). Previous studies demonstrated that just limited transcription factors (TF) were specifically expressed in embryonic TrkA+ nociceptive progenitor cells. The existence of a surprisingly small number of nociceptor-specific TFs implies that these TFs most likely play core roles in controlling nociceptor development. Our published works showed two of them, Runx1 and Tlx3, were co-expressed in most TrkA+ nociceptors at early embryonic stages. In conditional knockout mice, loss of Runx1 or Tlx3 function inhibited and decreased the expression of a range of sensory ion channels and receptors responding pain, itch and temperature. Furthermore, over-expression of these 2 TFs was able to ectopically induce an array of distinct nociceptive ion channels and receptors in mouse spinal cord, supporting our hypothesis that limited specific TFs are sufficient to determine the specification and differentiation of nociceptor, thermoceptor and pruriceptor. The research described here builds upon this preliminary work. The goal of our research over the next four years is to improve the transfection efficiency of different combination of nociceptor-specific TFs with viral vector, then directly induce human DRG-like nociceptors from skin fibrolasts with defined TFs in addition of morphogens. Characterization of nociceptors will include checking candidate gene expression profiles, microarrays and electrophysiology. This study will be helpful to clarify the molecular mechanism controlling the formation of a large cohort of nociceptors. Moreover, this work may lead to set up a novel analgesic drug screen platform based on induced human DRG-like nociceptors which may increase the chances of success to develop new analgesics.

把体细胞直接诱变成特异性的神经细胞是发育神经生物学的一个崭新的研究领域，这令获得某种神经疾病或某个病人特异性的神经细胞成为可能，进而可用于在体外建立疾病模型探讨致病机理及探寻治疗方法。文献报道及我们的研究显示仅有限的转录因子特异性地表达在TrkA+痛觉神经元前体中，包括Runx1和Tlx3。后续的工作证明这两个转录因子的功能缺失抑制了很多痛觉离子通道的生成，而过量表达Runx1和Tlx3可有效地在小鼠脊髓中直接诱导部分痛觉离子通道的产生。这暗示我们所筛选到的少数特异性转录因子即可决定痛相关感觉神经元的产生和分化。本课题在前期工作的基础上计划用病毒感染细胞的方法进一步提高转染不同组合转录因子的效率，再在外加不同生长因子的情况下直接诱导人皮肤成纤维细胞转变为痛相关感觉神经元。本研究将揭示多样性的痛相关感觉神经元产生的分子机理，并进而在诱导的人类痛相关神经元的基础上建立高效的止痛药物筛选平台。

胚胎早期背根神经节中TrkA+神经前体细胞逐渐分化成感知不同种类疼痛和瘙痒的多样性伤害感觉神经元，而其分化机制还不是完全清楚。文献报道及我们的研究显示仅有限的转录因子特异性地表达在TrkA+神经前体细胞中，预示着它们在伤害感觉神经元的发育过程中扮演着重要的角色。本课题经过大量的筛选工作证明转录因子Runx1、Tlx3、Ngn1、Islet1可以促进伤害感受神经元的体外诱导，并发现NGF是促进伤害感受神经元产生所必需的生长因子。进一步研究显示我们诱导的神经元可以应答痛觉离子通道TrpV1的激动剂辣椒素的刺激并产生动作电位。相比于对疼痛的研究，瘙痒的研究最近10年才有了比较大的进展，虽然部分瘙痒相关的受体、离子通道、神经递质以及神经元陆续被发现，但是关于这些分子的表达以及神经元发育的调控机制，目前来说还很不清楚。我们的研究显示转录因子Tlx3在瘙痒感受神经元的发育过程中起着重要的作用。首先，我们通过RNAseq的方法比较了Tlx3条件性敲除小鼠（Tlx3cko）和对照组小鼠背根神经节的转录组表达谱差异，发现Tlx3功能缺失导致大部分瘙痒相关分子的表达明显下降。mRNA原位杂交实验检测了部分瘙痒相关分子的表达，结果和RNAseq一致。行为学研究显示，Tlx3cko小鼠对多种致痒剂引起的急性瘙痒反应和对照组小鼠相比都明显减弱，说明Tlx3能够调控急性瘙痒行为的产生，既包括组胺依赖性瘙痒也包括非组胺依赖性瘙痒。另外，Tlx3也参与调控部分类型慢性瘙痒的产生，例如皮肤干燥。我们的研究证明虽然胚胎期转录因子Runx1表达是MrgprA3+和Nppb+瘙痒感受神经元产生所必需的，但在发育后期Runx1却对这两类瘙痒感受神经元的发育起到抑制作用，因此，出生后Runx1表达下调是此类瘙痒感受神经元能够继续正常发育的先决条件。胚胎早期TrkA+神经前体细胞逐渐向不同种类的伤害感觉神经元分化的过程中，瞬时表达Runx1的神经细胞分化成了瘙痒感觉神经元。本项目首次揭示了瘙痒感受神经元发育的分子机制，为今后瘙痒的治疗提供了理论依据。