丝氨酸蛋白酶抑制剂（SPINKs）家族在皮肤屏障功能中的作用机制

Dynamic coordination and balance between serine proteases and their specific inhibitors is important for the normal physiological function of skin in the body, such as normal physiological desquamation and epidermal barrier function. Disorder of dynamic balance may cause serious skin problems. For example, mutations in the SPINK5 gene can cause an autosomal recessive genetic disease Netherton syndrome and trigger atopic dermatitis (AD)-like lesions. While a wide range of serine proteases have been identified in the skin, there is little knowledge about their specific inhibitors. We have recently identified two novel members of the serine protease inhibitors kazal type (SPINKs). On this project, we aim to dissect the complete SPINKs family by the use of bioinformatics and molecular biology; to decipher molecular mechanisms involved in the function of newly discovered genes; to understand their important roles in skin barrier function as well as the correlation with AD by using techniques such as siRNA and knockout studies, etc. Meanwhile, in collaboration with Department of Dermatology at University Hospital Kiel, Germany, changes in the expression of these genes will be validated in the AD patient specimens and their role in the occurrence and development of AD lesions will be explored. We will also explore the SPINKs members as new players for diagnosis and potential therapeutic targets of AD patients.

丝氨酸蛋白酶与其特异抑制剂的动态协调平衡是维护人体皮肤正常生理功能的关键因素，其平衡失调可导致严重皮肤疾病的发生。例如, SPINK5基因的异常突变会引起人类皮肤常染色体隐性遗传疾病Netherton综合症，并触发异位性皮炎（atopic dermatitis，简称AD）样病变。然而，对应人体皮肤存在种类繁多的丝氨酸蛋白酶，目前对与之匹配的特异抑制剂的认识甚少。 在前期研究中，我们发现并坚定了Kazal型丝氨酸蛋白酶抑制剂家族（SPINKs）的两个新成员，预实验中又发现三个新成员。本项目将在此基础上采用生物信息学和分子生物学手段继续对SPINKs家族进行完整的分子鉴定， 明确新发现基因的表达调控机制和生化功能，并采用小分子siRNA、转基因及基因敲除等手段在细胞、组织和整体动物水平上研究其中重要成员在皮肤屏障功能缺陷中的作用及与AD的相关性，为AD等复杂性皮肤疾病的诊治提供新的思路。

皮肤屏障功能是维护人体健康抵御病菌侵染的第一道屏障，屏障功能的失调常常引发多种疾病。随着对Netherton综合征病变的发生与SPINK5基因相关性研究的积累，启发大家高度重视皮肤屏障功能对皮肤复杂性疾病发生发展的重要性。本项目旨在对SPINK基因家族，进行深层次的功能分析。1）对SPINK基因家族进行全面的基因结构鉴定，发现了三个新基因。2）对SPINK9的基因进行了全面的抗菌测定，明确了SPINK9通过与大肠杆菌伴侣蛋白SKP的互作，破坏膜的完整性进入细菌细胞质选择性杀灭大肠杆菌。这是我国在人体上发现的第一个天然抗菌肽,人源抗菌肽在替代抗生素的应用上具有很大的潜力。3）采用改良的CRISPR技术，构建成功SPINK6和SPINK14的基因敲除模型，并获得纯合子。进一步发现，SPINK6基因KO小鼠发生屏障功能受损，而SPINK14基因KO小鼠可能发生生殖障碍。这两个KO模型将会对今后深入研究皮肤屏障功能与皮肤复杂疾病/生殖系统的相关性产生重要的影响。