紫外线诱发光线性角化病中DNA-PKcs与Akt正反馈调控机制的研究

Ultraviolet (UV) induced DNA double strand breaks (DSBs) and signaling pathway activation are closely related to actinic keratosis (AK) occurrence. As the key enzyme in the repair of DSBs, DNA-PKCs inhibits injury induced apoptosis though excessive repair of DSBs, regulation of SP1 and c-fos activity in the nucleus, and complexs with SIN1 to activate Akt in the cytoplasm when is aberrantly activated; and DNA-PKCs abnormal activation is result from UV induced Akt transports into the nucleus and activates the former, thus forming a positive feedback regulation between DNA-PKCs and Akt, which may is an important mechanism for UV induced AK occurence. This subject intends to detection the activated level of DNA-PKCs, Akt and its downstream and related regulatory factors in AK lesions firstly, then to investigate UV induced DNA-PKCs, Akt cytoplasmic and nuclear transport, the order they are activated, the formation of DNA-PKcs/Akt complexes in the cytoplasm and nucleus, the effect on self-activation and downstream pathways activation, and the influences to DSBs repair, cell proliferation, cloning, apoptosis in cells level, and formation time, size, and number of AK lesions in mice level, to provide scientific data for clarifying the mechanisms of UV induced AK occurrence.

紫外线(UV)诱发的DNA双链断裂(DSBs)、增殖通路激活与光线性角化病(AK)密切相关。DNA-PKcs是修复DSBs关键酶，在其他组织中被异常激活后可在核内过度修复DSBs，并调控SP1、c-fos等活性，在胞浆中与SIN1结合激活Akt以阻碍损伤诱发的细胞凋亡；而DNA-PKcs的异常活化，与UV诱导Akt入核并激活前者相关，因此形成DNA-PKcs与Akt的正反馈调控机制，可能是UV诱发AK的重要机制。本课题拟从检测人AK皮损中DNA-PKcs、Akt及其下游和相关调控因子活化水平入手，在细胞和小鼠水平研究UV诱导DNA-PKcs、Akt的细胞质、细胞核转运，两者激活的先后顺序，DNA-PKcs/Akt复合物在细胞质、细胞核中的形成，对自身活化和下游通路的影响，及对DSBs修复、细胞增殖、克隆、凋亡等，和对小鼠AK形成时间、大小和数目等影响，为阐明UV诱发AK的机制提供科学数据。

紫外线(UV)诱发的DNA双链断裂(DSBs)、增殖通路激活与光线性角化病(AK)密切相关。DNA-PKcs是修复DSBs关键酶，在其他组织中被异常激活后可在核内过度修复DSBs，并调控SP1、cfos等活性，在胞浆中与SIN1结合激活Akt以阻碍损伤诱发的细胞凋亡；而DNA-PKcs的异常活化，与UV诱导Akt入核并激活前者相关，因此形成DNA-PKcs与Akt的正反馈调控机制，可能是UV诱发AK的重要机制。课题从检测AK组织中DNA-PKcs、Akt表达水平入手，在细胞和小鼠水平研究UV诱导DNA-PKcs/Akt之间的相互调控。结果显示：1.AK比正常组织、肿瘤细胞株比正常细胞株，DNA-PKcs和Akt均表达上调，磷酸化DNA-PKcs和Akt在胞核和胞浆中均增加；2.培养原代角质形成细胞，不同强度紫外线B(UVB)照射，检测发现DNA-PKcs主要位于胞核，随UVB照射时间延长在胞质中表达增加，而在胞核中表达减少；Akt主要位于胞质，总蛋白和磷酸化蛋白在胞质、胞核中随UVB照射时间延长表达增加。UVB照射后DSBs增加，但DNA-PKcs却从核内移至胞质，磷酸化Akt，并引发Akt向胞核移位。DNA-PKcs与Akt在胞核和胞质中，均有相互结合，UV照射后胞质中DNA-PKc/Akt的结合增加，且具有时间依赖性；3. DNA-PKcs和Akt干扰后，DNA-PKcs、Akt总蛋白和磷酸化蛋白在胞质、胞核中表达随UVB照射时间延长而增加，DNA-PKcs激活增加并转入胞浆，激活Akt信号通路，干扰DNA-PKcs时，Akt活化水平明显降低；4. UVB 照射后，原代角质形成细胞凋亡减少、增殖增加；5. UVB 照射敲除DNA-PKcs小鼠肿瘤形成数目更少，降低Akt、Akt Ser473磷酸化水平、Akt Thr308磷酸化水平、Bcl-2和转录因子SP1、c-fos、c-myc的表达水平，促进Bax、p53 serine-15的表达。综上所述，DNA-PKcs与Akt在肿瘤组织中表达增加，UVB照射促进DNA-PKcs从胞核移出，与Akt相互结合，促进Akt活化，导致细胞凋亡能力下降，增值活性增加。在敲除DNA-PKcs小鼠中发现UV照射后肿瘤形成数目更少，下游相关因子表达减少。证实了DNA-PKcs与Akt的正反馈调控机制，可能是UV诱发AK的重要机制。