核糖体蛋白L21基因（RPL21）突变对毛囊干细胞分化的影响及其机制

Hereditary hypotrichosis simplex (HHS) is an autosomal dominant form of congenital hair loss disorders characterized by generalized hair loss without any anomaly in other organ systems. Our previous studies have firstly revealed RPL21, which encodes ribosomal protein L21, as a new causative gene of HHS. And the mutation c.95G>A we reported in our study has been identified all the other HHS families with RPL21 mutations. Subsequently, we found that RPL21 expressed in the inner root sheath, outer root sheath and bulge area of the hair follicles where is the niche of hair follicle stem cells (HFSCs). RPL21 was shown to be related to the cell differentiation of cultured HaCaT cells, which implies RPL21 may play an important role in the regulation of differentiation of hair follicle cells. However, there is lack of further study on RPL21 over the world. RPL21 encodes a ribosomal protein that is a important component of the 60S subunit, ribosomal protein L21, which participates the process of potein synthesis. Some recent studies speculated that a small set of RPs exerts distinct or unique and tissue-specific functions on organismal development. Therefore, we are airm to establish and characterize an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. We will knockout RPL21 gene specifically and knock in RPL21 mutation c.95G>A within this cell model by CRISPR/Cas9 system. Then reveal the effects on the differentiation of HFSCs by RPL21 and the relationship between RPL21 and other hair follicle regulation signal pathways, especially Wnt/β-catenin pathway. This study is of great importance to the pathogenesis study of HHS. It could help us to reveal the role of RPL21 in the mechanism for control of hair growth and provide a new stratigy for the function study of tissue-specific ribosomal proteins.

遗传性单纯性少毛症是一种常染色体显性遗传的先天性秃发，累及全身毛发，而其它器官系统正常。本课题组首次发现RPL21为该病的新致病基因，且所有已知家系突变均为c.95G>A；我们还发现RPL21在毛囊内、外毛根鞘及毛囊干细胞所在的隆突部高表达，敲低RPL21表达影响HaCaT细胞的分化，提示RPL21可能参与调控毛囊的分化，但目前仍缺乏更深入的研究报道。RPL21编码核糖体60S大亚基组成蛋白，参与蛋白质合成，最新研究认为核糖体蛋白的突变对翻译功能的影响有器官特异性。因此本课题拟在前期研究基础上，建立永生化毛囊干细胞系，利用CRISPR/Cas9系统敲除RPL21和敲入点突变c.95G>A，研究RPL21对毛囊干细胞分化的影响及其与毛囊调控相关通路的关系。本研究对明确RPL21在毛囊干细胞分化过程中的作用有重要意义，为进一步明确HHS的致病机制及该病的治疗奠定理论基础。

遗传性单纯性少毛症是一种常染色体显性遗传的先天性秃发，累及全身毛发，而其它器官系统正常。我们前期研究发现RPL21为该病的新致病基因，且两个已报道家系突变均为c.95G>A。我们还发现RPL21在成人及胎儿头皮毛囊内、外毛根鞘及毛囊上皮干细胞所在的隆突部高表达，在人毛囊基质及毛囊乳头低表达。RPL21可能参与毛囊的生长调控和毛囊干细胞的分化。RPL21编码核糖体60S大亚基的组成蛋白，主要功能为参与蛋白质合成，核糖体蛋白的突变对翻译功能的影响有器官特异性。本研究基于C57BL/6N小鼠，利用CRISPR/Cas9技术建立了RPL21基因敲除及c.95G>A点突变小鼠动物模型，进一步对小鼠毛发表型进行鉴定并研究其对小鼠毛囊周期的影响。结果发现RPL21基因c.95G>A杂合点突变小鼠可发生毛发稀疏或斑秃样秃发表型，组织病理学存在真皮浅层瘢痕样改变及真皮深层噬黑素细胞等炎症后改变；在拔毛刺激诱导的同步化小鼠毛囊周期模型中，c.95G>A点突变小鼠较野生型小鼠的毛囊生长周期延迟，生长期滞后。RPL21主要表达于小鼠的毛囊内、外毛根鞘，在生长期毛球的毛基质及毛乳头无表达，表达部位与人类头皮毛囊相似。本研究还再次证实敲除RPL21基因可抑制HaCaT细胞的分化，并通过RNA-seq和蛋白质组学检测进一步分析RPL21影响HaCaT细胞分化的可能机制。此外，遗传性单纯性少毛症家系的突变研究中我们发现了新的家系存在c.95G>A点突变，进一步证实了RPL21基因突变的致病性。本研究对明确RPL21在毛囊生长调控中的作用具有重要价值，为进一步研究RPL21的功能和明确HHS的发病机制提供了重要的动物模型和研究基础。

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