Toll样受体3在p53调控下对中波紫外线照射的皮肤角质形成细胞免疫屏障损伤的修复机制研究

One of the effects of ultraviolet(UV) radiation on skin is breakdown of skin immune barrier mediated by UVB-induced immunosuppression. As a key component of the barrier, keratinocytes act as the first defense against UVB. It is of great importance fighting against photoedamaged skin disorders and cancers by repair mechanisms of keratinocyte immune barrier. With multiple functions that participating in innate immunity, photo immunity and skin barrier repair, Toll -like receptor 3 (TLR3), which functional expressed on keratinocytes, is an important target in research field of disruption of skin immune barrier caused by UV radiation. Our previous studies found that TLR3 signaling pathway can be activated by UVB in keratinocytes , while the expression of TLR3 is p53-dependent. We suppose that p53-dependent activation of TLR3 in keratinocytes may be involved in repair mechanisms of skin immune barrier destroyed by UVB radiation. In this study, keratinocytes culture, UVB radiation, intervention of p53 and TLR3 expressions, detections of DNA photoproducts, oxidative damage, immune defense components, immune suppression mediators, detection of target genes involved in skin barrier repair by custom PCR arrays and other research methods will be used. The disruption of keratinocyte immune barrier under UVB radiation will be observed. The repair of skin immune barrier by TLR3 will be revealed.The regulation of p53 on repair function of TLR3 will be analyzed. On the basis of studies above, the important role and key targets of p53-dependent activation of TLR3 in keratinocytes involved in repair mechanisms of skin immune barrier destroyed by UVB radiation will be elucidated, new ideas and novel approaches will consequently be provided for prevention and treatment of photodamaged skin disorders especially photocarcinogenesis.

UVB可诱导免疫抑制，破坏以角质形成细胞(KC)为重要组成的皮肤免疫屏障，导致光损性皮肤病和皮肤癌。Toll样受体3(TLR3)参与固有免疫、光免疫及屏障修复的多重功能，可能为光损伤皮肤免疫屏障修复的重要枢纽。我们前期研究发现UVB可激活KC的TLR3信号通路，其中TLR3的表达呈p53依赖性，故提出p53可能通过调控TLR3的表达，对UVB照射造成的KC免疫屏障破坏起到修复作用。本研究拟通过UVB照射、p53和TLR3表达的干预，DNA光产物、氧化损伤、免疫抑制介质、免疫防御成分的检测及皮肤屏障修复目的基因群检测等方法，研究UVB 照射对KC免疫屏障功能的影响、TLR3对光损下免疫屏障的修复作用、p53对TLR3修复屏障作用方式，深入揭示p53调控下TLR3对UVB诱导的KC免疫屏障损伤的修复作用机制，寻找出关键靶点，为光损性皮肤病包括光致皮肤癌的防治提供新思路。

本课题首先通过皮肤角质形成细胞（KC）的培养，研究UVB照射剂量和方式，建立了UVB诱导的KC免疫屏障损伤模型；然后探讨了UVB诱导KC免疫屏障损伤后细胞调控周期的变化，并探讨p53相关的生长停滞、细胞凋亡和肿瘤发生相关基因表达在UVB诱导的免疫屏障损伤中的变化情况；再通过UVB照射，以激动剂和抑制剂干预TLR3的表达，观察TLR3对DNA光产物产生、顺式尿刊酸含量、脂质过氧化、免疫抑制介质等损伤机制的抑制作用，观察TLR3对P53信号通路表达的影响，以及对固有免疫防御成分、屏障修复基因、固有免疫识别受体表达等皮肤免疫屏障防御功能的增强作用。.研究结果提示：一是建立了UVB诱导的KC免疫屏障损伤模型；二是研究发现UVB诱导的光损伤可激活KC的TLR3信号通路，后者不仅参与固有免疫应答，还参与皮肤屏障修复和维护，可能是光损伤后皮肤屏障功能修复机制的重要枢纽；三是研究发现TLR3激动剂可抑制UVB照射造成的TNF-α增加，可抑制后者的氧化损伤作用。TLR3激动剂可能通过激动TLR3来抑制UVB照射诱导产生的免疫抑制介质，有一定的免疫保护机制；四是研究结果支持了项目立项假设，即TLR3的表达呈p53依赖性和UVB剂量依赖性，证实了p53对TLR3通路及免疫保护机制存在调控作用，TLR3信号通路与p53介导的细胞凋亡、细胞衰老等光损伤修复、肿瘤抑制作用密切相关，p53通路下游的p21、bax可能是重要的调控靶点。上述结果初步证实了p53可调控 UVB照射下KC的TLR3的表达，并通过TLR3对光损伤造成的KC免疫屏障损伤起到修复作用，有可能为光损性皮肤病包括光致皮肤癌的防治提供一种新思路。.项目资助发表SCI论文2篇、中文核心论文3篇。培养博士生1名，硕士生1名（课题组成员），另外带教硕士生2名。获国家专利授权2项。