2023 年第 6 期 第 18 卷

基于高内涵成像分析系统筛选具有清除衰老细胞作用的天然产物

Screening of natural products with senescent cell eliminating effect based on high content imaging analysis system

作者：张聪聪王晓张燕红洪诗瑶杜杰

英文作者：Zhang Congcong Wang Xiao Zhang Yanhong Hong Shiyao Du Jie

单位：首都医科大学附属北京安贞医院北京市心肺血管疾病研究所心血管生物学研究室，北京100029

英文单位：Cardiovascular Biology Research Laboratory Beijing Anzhen Hospital Capital Medical University Beijing Institute of Heart Lung and Blood Vessel Diseases Beijing 100029 China

关键词：药物筛选；天然产物；衰老细胞清除剂；高内涵成像分析

英文关键词：Drugscreening；Naturalproducts；Senescentcellscavenger；Highcontentimaginganalysis

• 摘要：

• 目的  利用高内涵成像分析(HCS)系统筛选具有清除衰老细胞作用的天然产物。方法  将正常或使用阿霉素诱导衰老的小鼠3T3成纤维细胞接种于96孔板中。分别将45种天然产物（10 μmol/L）加至细胞培养基中刺激细胞4 d，使用HCS系统检测各孔中残留细胞数目，筛选出对正常细胞无影响但可清除衰老细胞的天然产物。并使用候选分子处理P53激动剂诱导衰老小鼠3T3成纤维细胞、阿霉素诱导的衰老人皮肤成纤维细胞或表皮细胞，检测残留细胞数目和细胞活性，以验证候选分子对各类衰老细胞的清除作用。结果  γ-生育三烯酚(γ-Toco)对正常细胞无影响的同时对小鼠衰老3T3成纤维细胞的杀伤作用最强。再验证结果显示γ-Toco的衰老细胞杀伤作用呈浓度依赖性(均P＜0.001)。10 μmol/L的γ-Toco可对P53激活剂诱导衰老的小鼠3T3成纤维细胞发挥杀伤作用，每个视野的细胞数明显少于二甲基亚砜对照处理［（33±6）个比（187±35）个］（P＜0.01）。10 μmol/L的γ-Toco也可杀伤衰老的人皮肤成纤维细胞和人表皮细胞(均P＜0.01)。结论  本研究基于HCS系统快速地筛选了一种可诱导衰老细胞死亡的天然产物，这种方法有利于清除衰老细胞分子的高效筛选平台的建立，从而加速抗衰老药物的研发。

• Objective    To screen natural products with senescent cell eliminating effect based on the high content imaging analysis system (HCS). Methods  Normal cells or doxorubicin induced senescent mouse 3T3 fibroblasts were cultured in 96 hole plate, and 45 natural products (10 μmol/L) were respectively added to cell culture to stimulate cells for 4 d. The HCS was used to detect the number of residual cells in each hole, screening out natural products that had no effect on normal cells but could clear senescent cells. The candidate molecules were also used to treat senescent mouse 3T3 fibroblasts induced by P53 activator, senescent human skin fibroblasts or epidermal cells induced by doxorubicin, and the number of residual cells and cellular activity were detected to verify the eliminating effect of candidate molecules on various senescent cells. Results  The γ-tocotrienol(γ-Toco) had the strongest killing effect on eliminating mouse senescent 3T3 fibroblasts while having no effect on normal cells. The revalidation results showed that the killing effect of γ-Toco on senescent cells was concentration dependence (all P＜0.001). The 10  μmol/L γ-Toco  could exert a killing effect on mouse 3T3 fibroblasts induced by P53 activator, and the number of cells in each field was significantly less than that in the dimethyl sulfoxide control treatment ［（33±6） vs （187±35）］（P＜0.01）. The 10 μmol/L γ-Toco could also kill the senescent human skin fibroblasts and epidermal cells (both  P＜0.01). Conclusion  This study quickly screened a natural product that can induce senescent cell death based on the HCS. This method is conducive to the establishment of an efficient screening platform for eliminating senescent cell molecules, thereby accelerating the development of anti-senescent drugs.