CPE对成纤维细胞衰老的影响及其分子机制研究
Study on Effect of CPE on Fibroblast Senescence and Its Molecular Mechanism

目的：探讨CPE的表达与细胞衰老的相关性。方法：选取接受手术清创的健康人供体(5~20岁)分离的人包皮的成纤维细胞(NHSF细胞)将其进行传代，在此我们定义“小于15代的NHSFs为N1组、15~25代的NHSFs为N2组、大于45代的NHSF为N3组”。随后用UVA和H₂O₂分别处理细胞使其衰老，建立早衰模型。此后进行CPE的慢病毒转染，获得CPE稳定敲除的细胞株。通过qRT-PCR和WB技术，检测CPE和衰老标志物蛋白及mRNA的表达。分别采用cck8和SA-β-gal染色检测CPE敲降后NHSFs的增殖和衰老程度。最后采用qRT-PCR检测CPE敲降后NHSFs促炎症因子的表达水平。结果：N1组CPE蛋白和mRNA表达最高，其次是N2组，N3组最少，早衰细胞模型中CPE蛋白和mRNA的表达减低，CPE敲降后NHSFs蛋白和mRNA的表达均升高、细胞活性降低、细胞衰老程度明显加快和促炎症因子mRNA表达升高。结论：CPE可作为潜在衰老标志物且其高表达可延缓衰老。
Objective: To explore the correlation between the expression of CPE and cellular senescence. Methods: Human foreskin fibroblasts (NHSF cells) isolated from healthy human donors (5~20 years old) who underwent surgical defibrillation were selected for passage. Here, we defined “NHSFs of less than 15 generations as group N1, NHSFs of 15~25 generations as group N2, and NHSF of more than 45 generations as group N3”. Then the cells were treated with UVA and H₂O₂ respectively to make them senescent, and the premature aging model was established. Then lentiviral transfection of CPE was performed to obtain stable CPE knockout cell lines. The expression of CPE and aging marker protein and mRNA were detected by qRT-PCR and WB. The proliferation and senescence of NHSFs after CPE knockdown were detected by cck8 and SA-β-gal staining, respectively. Finally, the expression level of NHSFs proinflammatory factors after CPE knock-down was detected by qRT-PCR. Results: The expressions of CPE protein and mRNA were the highest in N1 group, followed by N2 group and N3 group, and the expressions of CPE protein and mRNA were decreased in the progeria cell model. After CPE knockdown, the expressions of NHSFs protein and mRNA were both increased, the cell activity was decreased, the degree of cell senility was significantly accelerated, and the mRNA expression of proinflammatory factors was increased. Conclusion: CPE can be used as a potential senescence marker and its high expression can delay senescence.