TNF-α介导的角质形成细胞程序性坏死在SJS/TEN发病机制中作用的研究

Stevens-Johnson syndrome and toxic epidermal necrolysis are regarded as one spectrum of diseases with large area of skin exfoliation and severe mucosal involvement. Currently, it is a rare disease with high mortality due to no novel effective treatment. TNF-α monoclonal antibody achieved certain effect in recent small clinical application, but in this disease the exact mechanism of TNF-α is unclear, which restrict the expansion of these clinical drugs. It is widely accepted that keratinocytes ( KC) death is the central of the pathogenesis of this disease, including early apoptosis and and late necrosis. It is found in 2014 that KC necrosis of the disease is a new adjustable procedural necrosis named necroptosis. In some cell lines it has been found that TNF-α can initiate RIP3 protein kinase-mediated necroptosis when apoptosis pathway proteins especially caspase8 are blocked, but which is unknown in KC research. Our previous study found that high expression of TNF-α in this disease. Preliminary findings showed TNF-α alone could induce HaCaT apoptosis, when combined with the inhibitors of apoptosis in HaCaT cells TNF-α increased RIP3 expression, which accompanying with a large number of necrosis cells; we also preliminarily found decreased expression of caspase8 and increased RIP3 expression in the patient's skin tissue when divided as early and late samples. Based on the background, we propose the hypothesis: TNF-α can induce KC necroptosis in the disease when caspase8 activity decreased. We further carry out experiments to verify hypothesis through tissue, cells and animals, explore its mechanism and search for more precise therapeutic targets for clinical treatment.

Stevens-Johnson综合征及中毒性表皮坏死松解症是一组皮肤粘膜严重受累的疾病谱，罕见但死亡率高，临床无特效治疗。TNF-α单抗近来用于治疗本病取得一定的疗效，但它在本病中确切机制不明限制了此类药物的临床拓展。现认为角质形成细胞(KC)死亡是本病发病机制的中心环节，这一病理过程包括早期凋亡和后期坏死。最近发现本病KC坏死是一种新型的程序性坏死。一些细胞株中发现TNF-α在凋亡通路蛋白caspase8被抑制时可通过RIP3介导程序性坏死，但在KC中尚未有研究。我们前期初步发现TNF-α联合凋亡抑制剂处理HaCaT细胞可上调RIP3的表达，并出现大量细胞坏死；患者早、后期皮损中KC的caspase8表达下降而RIP3表达升高。据此我们推测TNF-α在caspase8活性下降时介导KC的程序性坏死。我们将从组织、细胞、动物三个方面开展实验验证，探索其机制，并为临床寻找更精确的治疗靶点。

Stevens-Johnson综合征(Stevens-Johnson syndrome, SJS)及中毒性表皮坏死松解症(toxic epidermal necrolysis, TEN)是一组以大面积表皮剥脱及粘膜严重受累的疾病。关于本病发病机制的研究主要围绕药物以各种途径导致角质形成细胞(Keratinocyte, KC)死亡展开。TEN/SJS 是以 KC 广泛性坏死为特征的疾病，程序性坏死在TEN中的作用机制尚缺乏研究。程序性坏死是一种可调控的胱天蛋白酶非依赖性细胞程序性死亡方式，细胞呈坏死样结构特点。在本研究中，从TEN组织、细胞、动物、三个层面进行实验。首先，从TEN患者红斑处取下皮损，电镜可观察到坏死及凋亡角质形成细胞。此外，免疫组化，实时荧光定量PCR法和蛋白质免疫印迹实验结果显示在分子水平上，红斑处皮损与非红斑处皮损比较，程序性坏死相关蛋白pRIP3,RIP3,pMLKL和MLKL均上调，caspase8下调。因此证明，在TEN患者红斑处皮损，程序性坏死被激活。实时荧光定量PCR法结果显示在分子水平上，红斑处皮损与非红斑处皮损比较，TNF-α、IFN-γ、FAS表达量升高。对TEN红斑皮损处T细胞进行培养，得到与正常皮肤相比，CD3+CD8+T细胞上调，在培养的上清液中用ELISA测得TNF-α、IFN-γ含量上升。用T细胞培养所得上清处理原代角质形成细胞，在蛋白水平验证程序性坏死相关蛋白pRIP3,RIP3,pMLKL和MLKL均上调，说明TNF-α、IFN-γ等细胞因子在TEN疾病进展中起作用。培养HaCaT 细胞及人原代角质形成细胞并重复实验，并验证TNF-α联合IFN-γ更易诱导人原代角质形成细胞发生程序性坏死。此外，应用Cre-loxP系统成功构建角蛋白14（KRT14）启动子控制的KC特异性caspase 8基因敲除小鼠模型Casp8flox/flox K14-Cre, 检测程序性坏死相关基因和蛋白表达量的差异，证实了TNF-α和INF-γ联合对Casp8表皮敲除的小鼠皮肤中程序性坏死产生的影响，以及Nec-1对TNF-α联合INF-γ在Casp8表皮敲除的小鼠皮肤中诱导的程序性坏死的抑制作用。研究结论将进一步理解本病的发病机制，并可能为目前临床治疗提供新的更精准的治疗靶点。