毛母质瘤致病基因鉴定及致病分子机制研究

Pilomatricoma, also named calcifying epithelioma, is an uncommon benign cutaneous tumor that mainly differentiates towards the hair matrix. Histological evaluation of 140,000 skin tumor samples yielded that pilomatricoma represented 0.12% of all the samples analyzed. Main features of clinicopathological characteristics about pilomatricomas: a large number of shadow cells in the center of lesion, infiltration of inflammatory cells with calcification in the interstitial tissue. Most pilomatricomas occur in the head and neck, usually present as an isolated lesion and sometimes undergo recurrence and malignant transformation, which put forward the necessary to reveal the etiology of this tumor. Up to now, only eleven small families with multiple familial pilomatricoma have been reported and no demonstrable underlying associations have been reported in the literature. The virulent gene and pathogenic mechanism for pilomatricoma are still not clear. Recently, a large family with multiple pilomatricoma was collected and presented as autosomal dominant inheritance. By exome sequencing, we found the first suspected pathogenic gene pl（c. G>A/ p. E>K）of pilomatricoma from germ line. The pl gene codes for PL protein, a member of PLC family. PL is abundantly expressed in the epidermis, required for both skin stem cell differentiation and homeostasis regulation of the immune system in skin. The purpose of current study is to uncover the function and the underlying pathogenic mechanism of PL mutation for pilomatricoma through analyses based on molecular level, cellular level and in vivo. Awareness of etiology for this lesion will be of great importance for its prevention, diagnosis and therapy.

毛母质瘤又名钙化上皮瘤，是一种以毛基质或毛母质分化异常为特点的罕见良性皮肤肿瘤。主要临床病理是病灶中心内见大量影子细胞，其间质内有炎症细胞浸润并伴有局部钙化形成。毛母质瘤多发于头颈部，直接影响容貌美观，且存在复发或恶变可能。现有文献只报道了11个小家系，所有家系均未进行遗传分析，毛母质瘤的致病基因及致病机制至今仍不清楚。本课题组近期收集了一个多发性毛母质瘤四代家系。利用外显子组测序技术，发现第一个毛母质瘤种系遗传可疑致病基因PL（c. G>A/ p. E>K），该基因编码PL蛋白，属于PLC家族。PL是表皮中PLC的主要类型，参与调控皮肤干细胞的分化过程及皮肤免疫系统的内稳态。本课题拟在分子、细胞和个体（小鼠模型）水平上研究PL突变体的功能，探讨PL突变如何影响细胞分化、引起细胞钙离子稳态失衡和组织炎症产生从而导致毛母质瘤发生，促进对毛母质瘤发生机制的认识，对其预防、诊断和治疗有重要意义。

毛母质瘤（Pilomatricoma，PM），是一种罕见的皮肤良性肿瘤，多发于头颈部，直接影响容貌美观，且存在复发或恶变可能。主要临床病理是病理特征表现为嗜碱性细胞-过渡细胞-影子细胞梯度变化，肿瘤中常有钙化累积。在散发性毛母质瘤病例瘤体细胞中检测出CTNNB1三号外显子突变，这些突变使β-catenin蛋白稳定性及入核量增加，细胞异常增殖而导致肿瘤发生，但是毛母质瘤的种系遗传基因及其致病机制尚不清楚。 .在该项目的资助下，收集4个PM家系和1例散发患者，对患者肿瘤组织进行详细病理分析，发现真皮内可见嗜碱性细胞和影细胞，过渡细胞细胞核逐渐消失，胞界清楚；抗凋亡标志物Bcl-2和毛囊分化标志物CD34表达水平随细胞层级变化而变化；细胞外豆腐渣样钙化沉积。对一个涉及32人8位患者PM大家系进行遗传分析，发现该疾病的第一个种系致病基因PLCD1 c.1186G>A（p.E396K）突变，体外功能分析表明该突变引起PLCδ1磷脂酶活性增强，并通过激活PKC/PKD/ERK1/2级联信号促进细胞增殖；PLCδ1-p.E396K突变引起过度分解PIP2，抑制细胞膜上的TRPV6钙通道活性而影响细胞的钙离子内流，使细胞外的钙离子积累产生钙化。体内研究，通过构建Plcd1q敲除和Plcd1E396K/E396K敲入小鼠模型，发现敲入小鼠皮肤Plcd1E396K/E396K小鼠皮肤组织中表皮分化标志基因krt10，flg，lor和ivl表达下调，而增殖标志基因p63和ccnd1表达上调；Plcd1E396K/E396K原代皮肤角质细胞增殖活性比野生型更强；8月龄的Plcd1E396K/E396K小鼠腹部皮肤出现自发肿瘤，肿瘤内存在大量核皱缩细胞、无核细胞和明显的钙化累积，抗凋亡标志物bcl-2和毛囊分化标志物CD34表达模式与人类毛母质瘤相似。该研究首次揭示来自种系突变导致多发毛母质瘤的致病机理，对其预防、诊断和治疗PM有重要意义。.该项目实施过程中参加12人次国内学术交流会议，在该项目支持下，发表标注该项目SCI论文10篇。培养博士生9名，硕士生2名和3位博士后。

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