线粒体DNA启动细胞免疫应答介导感光细胞丢失的机制研究

Retinal degeneration is a general term for a series of retinal degeneration or dystrophy diseases. It is also a common outcome of many retinal diseases. Progressive photoreceptor cell loss is a common pathological feature. The immune response and immune damage of retinal photoreceptor cells are the main causes of cell loss, but the specific molecular mechanisms are still unclear. Current researches focused on the immune response initiated by autologous DNA which was recognized by cytoplasmic DNA sensing system. The DNA, which is exposed to the cytoplasm, is sensed by cGAS and initiates the immune response through the cGAMP/STING signaling pathway. Our previous studies have found that the escape of mitochondrial DNA to the cytoplasm is a common phenomenon in photoreceptor cells. Therefore, we speculate that the loss of photoreceptor cells in retinal degeneration is mediated by abnormal exposure mtDNA in the cytoplasm priming immune damage. Glucocorticoid-Induced Leucine Zipper (GILZ) has a "leucine zipper", which can specifically combine with DNA to form a DNA- protein complex. Our previous study confirmed that GILZ could inhibit retinal inflammation and neuron protection. Therefore, we further speculate that GILZ could interact with mtDNA and inhibit immune response initiated by mtDNA. In order to confirm those hypothesize, the SD rats and photoreceptor cells were stimulated with autologous mitochondrial DNA respectively, the activation of cGAS-cGAMP-STING pathway and downstream molecular detection by PCR, Western blot and immunofluorescence. The immunoprecipitation was used to detect interaction of the cGAS or GILZ with mtDNA, respectively.

全球约50%的致盲性眼病由感光细胞丢失导致，但丢失的机制仍然不明确。异常暴露的自体DNA介导的细胞损伤是当前研究热点，而线粒体是自体DNA的重要来源，高度保守的线粒体DNA(mtDNA)逃逸后可启动细胞免疫应答介导细胞损伤。我们前期已证实感光细胞mtDNA逃逸现象，但逃逸的mtDNA是否介导感光细胞损伤及损伤机制目前仍不清楚。激素诱导锌指状蛋白（GILZ）是具有“亮氨酸拉链”结构蛋白，其能抑制视网膜炎症反应、保护光细胞丢失，其亮氨酸拉链能结合DNA并形成DNA-蛋白复合物。因此我们推测病理刺激诱导感光细胞mtDNA逃逸，后者被位于胞浆内DNA感知分子识别，然后介导细胞免疫应答及损伤。同时，GILZ竞争性结合逃逸的mtDNA，抑制mtDNA的免疫启动，从而发挥免疫调节及细胞保护功能。本课题拟阐明自体逃逸的mtDNA诱导感光细胞丢失的具体分子机制，同时我们也将阐明GILZ保护感光细胞机制。

目的：探索mtDNA诱导感光细胞损伤的机制.方法：脂多糖、过氧化氢、光损伤以及高糖等多种手段刺激视网膜血管内皮细胞以及661W细胞， 用PCR技术分别对胞浆内及线粒体内mtDNA进行定量，采用MitroTracker Red和Pico green进行mtDNA定位；线粒体通道转换孔（mPTP）试剂盒检测细胞mPTP的开放情况。然后我们体外提取mtDNA，然后采用质粒转染的方法外源性植入mtDNA至细胞或者视网膜组织的胞浆内，采用以下方法探索mtDNA的损伤机制：采用免疫荧光技术定位STING的位置；WB技术检测TBK1、IRF3、NF-kB p65等的磷酸化情况，同时分别检测细胞核及胞浆内p-IRF3和p-P65的量；用WB及RT-PCR技术分别检测Bak/Bax的转录及翻译，检测细胞因子CCL4，CXCL10，IRF1，IFNB1和ICAM1的转录及翻译；用WB技术检测线粒体内及胞浆内cyto C的量以及caspase9/3的级联激活情况；用TUNEL技术检测感光细胞凋亡情况；用ERG检测大鼠视网膜功能。.结论：病理刺激诱导mtDNA释放至胞浆内是一种普遍病理现象，病理刺激后用过开放线粒体通道转换孔（mPTP）释放mtDNA至胞浆内。异常暴露于胞浆内的mtDNA被位于胞浆内的DNA感知元件cGAS所感知，cGAS识别胞浆的mtDNA片段后，反馈上调细胞内cGAS的合成，cGAS可加速合成cGAMP，合成的cGAMP作为二级信号分子与与 stimulator of type I IFN gene（STING）结合并激活 STING，活化的STING从高尔基体上游离并转位至细胞核周围，位于细胞核周围的STING可以通过多条信号通路发挥作用，一方面激活TBK1-IRF3信号通路，磷酸化的IRF3，同时还促进NF-kB p65磷酸化，磷酸化的IRF3及磷酸化的NF-kB P65作为细胞内的重要核转录因子，促进Bak/Bax转录及翻译，上调的Bak/Bax在线粒体膜表面形成一个通道，释放位于线粒体内的cyto C至胞浆内，胞浆内的cyto C进一步级联激活caspase9/3，诱导视网膜感光细胞凋亡，导致大鼠视网膜功能异常；同时磷酸化的核转录因子还促进多种细胞因子的转录及翻译，其中包括CCL4，CXCL10，IRF1，IFNB1和ICAM1，导致视网膜细胞及视网膜组织免疫炎