Saldías María Paz, Fernández Christian, Morgan Alejandra, Díaz Catalina, Morales Diego, Jaña Fabián, Gómez Alvaro, Silva Alonso, Briceño Fernanda, Oyarzún Alejandro, Maldonado Felipe, Cerda Oscar, Smith Patricio C, Cáceres Mónica
Program of Molecular and Cell Biology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile.
Universidad de Aysén, Coyhaique, Chile.
PLoS One. 2017 Sep 12;12(9):e0184189. doi: 10.1371/journal.pone.0184189. eCollection 2017.
Aging is a gradual biological process characterized by a decrease in cell and organism functions. Gingival wound healing is one of the impaired processes found in old rats. Here, we studied the in vivo wound healing process using a gingival repair rat model and an in vitro model using human gingival fibroblast for cellular responses associated to wound healing. To do that, we evaluated cell proliferation of both epithelial and connective tissue cells in gingival wounds and found decreased of Ki67 nuclear staining in old rats when compared to their young counterparts. We next evaluated cellular responses of primary gingival fibroblast obtained from young subjects in the presence human blood serum of individuals of different ages. Eighteen to sixty five years old masculine donors were classified into 3 groups: "young" from 18 to 22 years old, "middle-aged" from 30 to 48 years old and "aged" over 50 years old. Cell proliferation, measured through immunofluorescence for Ki67 and flow cytometry for DNA content, was decreased when middle-aged and aged serum was added to gingival fibroblast compared to young serum. Myofibroblastic differentiation, measured through alpha-smooth muscle actin (α-SMA), was stimulated with young but not middle-aged or aged serum both the protein levels and incorporation of α-SMA into actin stress fibers. High levels of PDGF, VEGF, IL-6R were detected in blood serum from young subjects when compared to middle-aged and aged donors. In addition, the pro-inflammatory cytokines MCP-1 and TNF were increased in the serum of aged donors. In old rat wound there is an increased of staining for TNF compared to young wound. Moreover, healthy gingiva (non injury) shows less staining compared to a wound site, suggesting a role in wound healing. Moreover, serum from middle-aged and aged donors was able to stimulate cellular senescence in young cells as determined by the expression of senescence associated beta-galactosidase and histone H2A.X phosphorylated at Ser139. Moreover, we detected an increased frequency of γ-H2A.X-positive cells in aged rat gingival tissues. The present study suggests that serum factors present in middle-aged and aged individuals may be responsible, at least in part, for the altered responses observed during wound healing in aging.
衰老 是一个渐进的生物学过程，其特征是细胞和机体功能下降。牙龈伤口愈合是老年大鼠中受损的过程之一。在这里，我们使用牙龈修复大鼠模型研究了体内伤口愈合过程，并使用人牙龈成纤维细胞建立体外模型来研究与伤口愈合相关的细胞反应。为此，我们评估了牙龈伤口中上皮和结缔组织细胞的增殖情况，发现与年轻大鼠相比，老年大鼠的Ki67核染色减少。接下来，我们评估了从年轻受试者获取的原代牙龈成纤维细胞在不同年龄个体的人血清存在下的细胞反应。18至65岁的男性捐赠者被分为3组：18至22岁的“年轻组”、30至48岁的“中年组”和50岁以上的“老年组”。与年轻血清相比，当向牙龈成纤维细胞中添加中年和老年血清时，通过Ki67免疫荧光和DNA含量流式细胞术测量的细胞增殖减少。通过α-平滑肌肌动蛋白（α-SMA）测量的肌成纤维细胞分化，在添加年轻血清时受到刺激，而添加中年或老年血清时则未受到刺激，无论是蛋白质水平还是α-SMA掺入肌动蛋白应力纤维的情况。与中年和老年捐赠者相比，年轻受试者血清中检测到高水平的血小板衍生生长因子（PDGF）、血管内皮生长因子（VEGF）、白细胞介素-6受体（IL-6R）。此外，老年捐赠者血清中促炎细胞因子单核细胞趋化蛋白-1（MCP-1）和肿瘤坏死因子（TNF）增加。与年轻伤口相比，老年大鼠伤口中TNF染色增加。此外，健康牙龈（未损伤）与伤口部位相比染色较少，表明其在伤口愈合中起作用。此外，如通过衰老相关β-半乳糖苷酶的表达和在丝氨酸139处磷酸化的组蛋白H2A.X所确定的，中年和老年捐赠者的血清能够刺激年轻细胞的细胞衰老。此外，我们在老年大鼠牙龈组织中检测到γ-H2A.X阳性细胞的频率增加。本研究表明，中年和老年个体中存在的血清因子可能至少部分负责衰老过程中伤口愈合期间观察到的反应改变。
