Yu Xing, Quan Jing, Chen Shuai, Yang Xinyue, Huang Shuai, Yang Gang, Zhang Yujing
Key Laboratory of Molecular Epidemiology of Hunan Province, School of Medicine, Hunan Normal University, Changsha, China.
Key Laboratory of Model Animals and Stem Cell Biology of Hunan Province, School of Medicine, Hunan Normal University, Changsha, China.
Front Integr Neurosci. 2022 Jul 29;16:929788. doi: 10.3389/fnint.2022.929788. eCollection 2022.
Aging may be the largest factor for a variety of chronic diseases that influence survival, independence, and wellbeing. Evidence suggests that aging could be thought of as the modifiable risk factor to delay or alleviate age-related conditions as a group by regulating essential aging mechanisms. One such mechanism is cellular senescence, which is a special form of most cells that are present as permanent cell cycle arrest, apoptosis resistance, expression of anti-proliferative molecules, acquisition of pro-inflammatory, senescence-associated secretory phenotype (SASP), and others. Most cells cultured or may undergo cellular senescence after accruing a set number of cell divisions or provoked by excessive endogenous and exogenous stress or damage. Senescent cells occur throughout life and play a vital role in various physiological and pathological processes such as embryogenesis, wound healing, host immunity, and tumor suppression. In contrast to the beneficial senescent processes, the accumulation of senescent also has deleterious effects. These non-proliferating cells lead to the decrease of regenerative potential or functions of tissues, inflammation, and other aging-associated diseases because of the change of tissue microenvironment with the acquisition of SASP. While it is understood that age-related diseases occur at the cellular level from the cellular senescence, the mechanisms of cellular senescence in age-related disease progression remain largely unknown. Simplified and rapid models such as models of the cellular senescence are critically needed to deconvolute mechanisms of age-related diseases. Here, we obtained replicative senescent L02 hepatocytes by culturing the cells for 20 weeks. Then, the conditioned medium containing supernatant from replicative senescent L02 hepatocytes was used to induce cellular senescence, which could rapidly induce hepatocytes into senescence. In addition, different methods were used to validate senescence, including senescence-associated β-galactosidase (SA-β-gal), the rate of DNA synthesis using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and senescence-related proteins. At last, we provide example results and discuss further applications of the protocol.
衰老可能是影响生存、独立性和幸福感的多种慢性疾病的最大因素。有证据表明，衰老可被视为一个可调节的风险因素，通过调控关键的衰老机制来延迟或缓解与年龄相关的一系列状况。细胞衰老就是这样一种机制，它是大多数细胞的一种特殊形式，表现为永久性细胞周期停滞、抗凋亡、抗增殖分子表达、获得促炎的衰老相关分泌表型（SASP）等。大多数培养的细胞或在经历一定次数的细胞分裂后，或受到过多内源性和外源性应激或损伤的刺激，可能会发生细胞衰老。衰老细胞在整个生命过程中都会出现，并在胚胎发育、伤口愈合、宿主免疫和肿瘤抑制等各种生理和病理过程中发挥重要作用。与有益的衰老过程相反，衰老细胞的积累也有有害影响。这些不增殖的细胞由于随着SASP的获得导致组织微环境改变，从而导致组织再生潜能或功能下降、炎症以及其他与衰老相关的疾病。虽然人们知道与年龄相关的疾病是在细胞水平上由细胞衰老引起的，但细胞衰老在与年龄相关疾病进展中的机制仍 largely未知。迫切需要如细胞衰老模型这样简化且快速的模型来解析与年龄相关疾病的机制。在此，我们通过将细胞培养20周获得了复制性衰老的L02肝细胞。然后，用含有复制性衰老L02肝细胞上清液的条件培养基来诱导细胞衰老，其可迅速诱导肝细胞衰老。此外，还使用了不同方法来验证衰老，包括衰老相关β半乳糖苷酶（SA-β-gal）、使用5-乙炔基-2'-脱氧尿苷（EdU）掺入法检测DNA合成速率以及衰老相关蛋白。最后，我们给出了示例结果并讨论了该方案的进一步应用。
