IL-24调控维持上皮细胞分化稳态的作用及机制研究

Many diseases are related with the disorder of epithelial differentiation and homeostasis in human. To date, the effect and mechanism of interleukin 24 (IL-24) on epithelial differentiation and homeostasis are still unreported. In this proposal, based on our preliminary results, we initially deduced that Il-24 could play a very important role in the maintenance of the epithelial differentiation and homeostasis. Meanwhile, we advanced a "pendulum swing theory" to explain how IL-24 works on regulation of epithelial differentiation and homeostasis. That is, IL-24 can maintain the homeostasis of epithelium. Both low-differentiated and high -differentiated epithelial cells can differentiate into the middle differentiated status of epithelium under IL-24 treatment. We therefore design the experiments at cell level, at three-dimensional epithelial cell cultivation level, and at the IL-24 transgenic and knockout mice level, through cell culture and treatment with IL-24 method to study the effect of IL-24 on the differentiation of epithelial cells HaCat, oral squaumous cells WSU-HN6, and embryo stem cells; establishment IL-24 receptor transfected stable cell line to investigate the model of action of IL-24, using real time PCR, western blot, immunoprecipitation and immunostaining to study the expression of epithelial differentiation markers and the signal transduction involved; using luciferase to explore the regulation of IL24 at DNA level; three-dimensional epithelial cell culture and the skin tissue specimens from IL-24 transgenic and knockout mice to confirm the data obtained. Our final goal is to investigate and understand the effect of IL-24 on epithelial differentiation and how Il-24 works, the underlying mechanism.

人类许多疾病都与上皮细胞的分化和稳态有关，研究调控上皮稳态调控机制对某些疾病的预防和治疗有重要意义。目前,白介素24（IL-24）对调控上皮分化和稳态的影响及其机理尚无报道。本课题根据我们的前期研究结果，初步认定IL-24对调控上皮分化和稳态起重要的作用，并提出了IL-24对上皮细胞分化及维持稳态的"钟摆理论"，即IL-24可以维持上皮细胞分化稳态，低分化及过分化上皮细胞在IL-24 的作用下，均可以维持在特定的中间状态分化。为此我们在细胞层面、上皮三维培养层面及IL-24转基因及敲除动物上皮标本层面，拟通过IL-24蛋白刺激上皮细胞、建立表达IL-24受体的口腔上皮细胞系，Real time PCR、Western blot、荧光素酶实验、免疫共沉淀、上皮三维培养和IL-24转基因和敲除动物上皮组织标本分析上皮分化程度等技术，来探讨IL-24调控维持上皮分化和稳态中的作用及调控机理。

人类许多疾病都与上皮细胞的分化和稳态有关，研究调控上皮稳态调控机制对某些疾病的预防和治疗有重要意义。通过本项目研究，我们在细胞层面，初步明确了IL-24对上皮分化的影响，证实了本课题的科学假设，即IL-24对上皮细胞分化及维持稳态的"钟摆理论"。IL-24可以维持上皮细胞分化稳态，低分化及过分化上皮细胞在IL-24 的作用下，均可以维持在特定的中间状态分化。经rhIL24处理后，HaCaT与HN6-IL20R2细胞的p-STAT3分子都处于激活状态；与维持上皮层基底细胞干性有关的p63α（这里的抗体检测包括TAp63和ΔNp63）表达都增高；但与上皮分化状态相关的Notch1和Ap2α表达在HaCaT与HN6-IL20R2细胞中却有着相反的表达模式。HaCaT细胞在rhIL24作用后，Notch1表达降低，在HN6-IL020R2细胞系中，Notch1表达升高。这种规律在Ap2α也同样存在。Notch1和Ap2α的表达变化趋势与细胞终末分化标记物的变化趋势一致，提示Notch1和Ap2α与IL-24调控上皮分化和稳态有关。现已初步证实了IL-24通过Notch1和Ap2α调控上皮分化和稳态，并且这种调控可能与甲基化转移酶DNMTs相关。在研究过程中，我们探索了体外构建上皮三维培养的方法，为下一步改良和利用此模型进行上皮分化研究提供了经验和实验基础。在转基因动物层面，我们成功构建了MDA-7/IL-24基因敲除的动物模型。在转化研究方面，我们利用IL-24具有抗肿瘤特性及双歧杆菌的靶向性，增加了双歧杆菌介导的IL-24中作为新型治疗载体的研发工作。现已成功构建携带该基因的体内靶向载体，并验证了该系统的靶向性及抑癌作用。在本项目的后期实验设计中，我们打算利用课题组前期工作中的远红外蛋白mKate2的表达来实验靶向性的实时追踪。本项目研究成果的价值在于提出IL-24调控上皮分化和稳态的新机制，并为下一步利用双歧杆菌介导的IL-24作为治疗肿瘤的新型载体及可能的临床转化做了有益的探索。