Acra Alejandro Mena, Sakagami Hiroshi, Uota Shin, Yoshihara Masaaki, Kito Shinji, Izawa Maki, Ohtaka Yusei, Nakaya Giichirou, Koga-Ogawa Yukari, Nobesawa Tadamasa, Ueda Daisuke, Suzuki Ryuichiro
Meikai University Research Institute of Odontology (M-RIO), Saitama, Japan.
Faculty of Dentistry, Autonomous University of the State of Mexico (UAEMex), Toluca, Mexico.
In Vivo. 2025 Sep-Oct;39(5):2534-2548. doi: 10.21873/invivo.14055.
BACKGROUND/AIM: With an increasing number of comorbid diseases, the number of drug intake by elderly people and their chances to X-ray exposure inevitably increase. However, it is unclear how these treatments affect longevity. Primary human diploid fibroblasts with limited life span have been used as a model system for the study of cellular senescence. Since aging is a long process, the colony formation assay has often been used to monitor the aging progression. However, this method cannot accurately quantitate the anti-aging activity of the test samples due to the heterogeneous size distribution of the colonies. Therefore, we developed a new "overlay" method for quantitating the replicative lifespan elongation (RLE) activity of substances of interest.
Cells were treated for 14 days with or without (as control) various concentrations of test samples in culture medium supplemented with fetal bovine serum (FBS), without medium changes, but overlayed with fresh medium to minimize the cell detachment and nutritional deprivation. From the dose-response curve, the elongation index of the survival time (EI) was calculated by the ratio of maximum viable cell number at the optimal concentration of the test sample to that of the control at Day 14.
In general, six human fibroblasts prepared from dermal, oral and lung tissues required higher concentrations of FBS than cancer cells. Dermal fibroblasts were selected as the target cells, based on their higher hormetic responses. Quercetin, hydrocortisone, sodium ascorbate, vanillic acid and vanillin showed higher EI values than estradiol, curcumin, resveratrol, phellamurin, sodium butylate and X-ray irradiation, but did not reach the levels of plant extracts.
The present method may be useful to quantitate the RLE activity of external and internal factors alone or in combination, in the presence of an appropriate positive control.
背景/目的：随着合并症数量的增加，老年人的药物摄入量及其接受X射线照射的机会不可避免地增多。然而，尚不清楚这些治疗如何影响寿命。有限寿命的原代人二倍体成纤维细胞已被用作细胞衰老研究的模型系统。由于衰老过程漫长，集落形成试验常被用于监测衰老进程。然而，由于集落大小分布不均一，该方法无法准确量化测试样品的抗衰老活性。因此，我们开发了一种新的“覆盖”方法来定量感兴趣物质的复制寿命延长（RLE）活性。
在补充有胎牛血清（FBS）的培养基中，用不同浓度的测试样品处理细胞14天，有或无（作为对照），不更换培养基，但覆盖新鲜培养基以尽量减少细胞脱离和营养剥夺。根据剂量反应曲线，通过测试样品最佳浓度下第14天的最大活细胞数与对照的最大活细胞数之比计算存活时间延长指数（EI）。
一般来说，从皮肤、口腔和肺组织制备的六种人成纤维细胞比癌细胞需要更高浓度的FBS。基于其更高的应激反应，选择皮肤成纤维细胞作为靶细胞。槲皮素、氢化可的松、抗坏血酸钠、香草酸和香草醛的EI值高于雌二醇、姜黄素、白藜芦醇、桑皮苷、丁酸钠和X射线照射，但未达到植物提取物的水平。
在有适当阳性对照的情况下，本方法可能有助于单独或联合定量内外部因素的RLE活性。
