目的： 反常性痤疮（acne inversa, AI）又称作化脓性汗腺炎（Hidradenitis suppurativa，HS），是严重影响患者身心健康的慢性炎症性皮肤病，其发病机制尚未明确。编码γ-分泌酶组分的基因的失功能性变异，特别是NCSTN基因的变异，与家族性AI病人高度相关。AI病人中，表皮细胞的增殖水平升高且分化异常，而转化生长因(transforming growth factor, TGF)-β的水平降低。但是，TGF-β信号通路激活降低是否参与到以上过程尚且未知。本研究的目的为探究敲降NCSTN基因后人永生化角质形成细胞HaCaT中TGF-β通路激活水平的变化，及其在人永生化角质形成细胞增殖和分化中的作用。 方法： 应用本实验室冻存的敲降NCSTN的HaCaT细胞（以下称为“shRNA组”）进行如下实验。通过CCK８实验来研究细胞的增殖曲线。通过qPCR实验探究感兴趣基因的mRNA表达水平。通过Western blotting实验来验证候选蛋白质的表达，包括磷酸化的蛋白质的表达。应用TGF-β通路新型的激动剂SRI-011381来激活培养的细胞中的TGF-β通路，以探究TGF-β通路对shRNA组细胞增殖和分化的影响。 结果： １.　shRNA组HaCaT细胞中NCSTN的表达的mRNA水平和蛋白质水平均较对照组NC下降，表明此为高质量的实验体系。 2. shRNA组HaCaT细胞中TGF-β通路中的成员的mRNA表达水平显著降低。 3. shRNA组HaCaT细胞中TGF-β通路中的主要配体，TGF-β1的蛋白质水平降低；磷酸化的SMAD2和SMAD3水平降低；而抑制性的SMAD7表达增加。TGFβ通路的激活水平降低。 4. 在shRNA组HaCaT细胞中应用TGF-β通路的激活剂SRI-011381逆转细胞增快的生长速度，并降低Ki-67和PCNA的表达。 5. 在shRNA组HaCaT细胞中应用SRI-011381激活TGF-β通路并不改变异常的分化相关蛋白质的表达。 结论： NCSTN基因的敲降导致人永生化角质形成细胞中TGF-β通路活性的降低，后者进一步导致细胞增殖水平的升高。在AI病人中进行TGF-β激活的治疗可能是未来对AI有效的治疗手段。

Aims: Patients with acne inversa (AI), which is a chronic inflammatory skin disease, suffer from symptoms of the disorder as well as loss of quality of life. The precise mechanisms of AI have not been known. Loss-of-function mutations of genes encoding components in γ-secretase, including NCSTN have been shown to be positively associated with family AI. In AI patients, epidermal cells have increased levels of proliferation and abnormal differentiation, whereas activation of the transforming growth factor (TGF)-β signaling pathway decreases. However, whether TGF-β signaling is involved in the changes remains unknown. The aim of this project is to test whether TGF-β signaling decreases in HaCaT cells with NCSTN knockdown, and roles of this signaling pathway in proliferation and differentiation NCSTN knockdown HaCaT cells Methods: HaCaT cells with NCSTN knockdown (referred to as shRNA cells) were used in this study. Proliferation was measured using the CCK8 assay. mRNA levels of genes of interest were assessed using q-PCR. Protein levels and phosphorylation of target proteins were measured by Western blotting. Activation of TGF-β signaling was achieved by application of SRI-011381 in cultured cells. Results: 1.shRNA cells had decreased levels of NCSTN in both mRNA and protein levels. 2.mRNA levels of genes in the TGF-β signaling pathway decreased. 3.Protein levels of TGF-β1, a key ligand in the TGF-β signaling pathway, decreased in shRNA cells. Levels of phosphorylated SMAD2 and SMAD 3 decreased, and inhibitory SMAD7 increased in shRNA cells. Activation of the TGF-β signaling pathway was downregulated in shRNA cells. 4.Application of SRI-011381 reversed increased proliferation in shRNA cells, and decreased mRNA expression of Ki-67 and PCNA. 5.No effects were observed in differentiation of shRNA cells treated with SRI-011381. Conclusion: Knockdown of NCSTN in HaCaT cells causes decreased activation of TGF-β signaling, which consequently induces increased proliferation. Activation of TGF-β signaling might be a viable therapeutic option for those with AI.