健骨二仙丸对氧化应激致衰老骨髓间充质干细胞增殖、凋亡及p16INK4、p21CIPI、p53表达的影响*
作者：郭柏铭1，刘文松1，孙 丹1，谢秋雯1，洪 莉1，陈小香1，程志安2
单位：1.广州白云山医院，广东 广州 510515； 2.广东省中医院，广东 广州 510120
引用：引用：郭柏铭，刘文松，孙丹，谢秋雯，洪莉，陈小香，程志安.健骨二仙丸对氧化应激致衰老骨髓间充质干细胞增殖、凋亡及p16INK4、p21CIPI、p53表达的影响[J].中医药导报,2025,31(11):14-19.
DOI：10.13862/j.cn43-1446/r.2025.11.003
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摘要：目的：研究健骨二仙丸含药血清对大鼠骨髓间充质干细胞（BMSCs）氧化应激损伤衰老后的细胞代谢活力、凋亡及衰老相关基因表达的影响。方法:采用全骨髓贴壁法培养BMSCs；流式细胞仪检测BMSCs表面标记物CD29、CD34、CD44、CD45、CD90。采用传统造模剂H2O2，以浓度500 μmol/L H2O2处理BMSCs 24 h诱导细胞氧化应激衰老模型建立；β-半乳糖苷酶染色确定BMSCs衰老模型的成功建立；健骨二仙丸含药血清干预氧化损伤衰老的BMSCs，采用MTT法分别检测健骨二仙丸含药血清对氧化衰老BMSCs 24、48、72 h后代谢活力的影响；运用流式细胞仪技术检测药物作用于衰老BMSCs 24 h后细胞凋亡情况；以反转录荧光定量技术（RT-q real-time PCR）观察衰老相关基因p16INK4、p21CIPI、p53表达水平的变化。结果：通过应用0.5 mmol/L浓度的H2O2作用24 h后可使BMSCs在形态学上有衰老的改变，β-半乳糖苷酶染色阳性率大于90%；以5%浓度健骨二仙丸含药血清作用衰老的BMSCs 24、48 h和10%药物浓度作用24 h后，可促进细胞增殖（P<0.05），而20%药物浓度作用BMSCs 48 h有抑制其增殖的作用（P<0.05）；以10%含药血清浓度作用老化的BMSCs 24 h可抑制细胞凋亡（P<0.05），下调衰老相关基因p21CIPI与p53的表达（P<0.05），而p16INK4的差异无统计学意义（P>0.05）。结论:健骨二仙丸对氧化衰老BMSCs有明显促进增殖，增强大鼠BMSCs的代谢，其凋亡率明显降低，在衰老相关基因p21CIPI、p53表达方面也达到一定调节作用，发挥抗氧化衰老作用是通过多种途径完成的。
关键词：健骨二仙丸；氧化应激；衰老；骨髓间充质干细胞；增殖；凋亡
Abstract：
Objective: To investigate the effects of serum containing Jiangu Erxian pills on metabolic activity, apoptosis, and the expression of senescence-associated genes in rat bone marrow mesenchymal stem cells (BMSCs) after oxidative stress-induced senescence. Methods: BMSCs were cultured using the whole bone marrow wall method. Flow cytometry was used to detect surface markers CD29, CD34, CD44, CD45 and CD90 on BMSCs. An oxidative stress-induced senescence model was established by treating BMSCs with 500 μmol/L H2O2 for 24 hours. Successful establishment of the BMSCs senescence model was confirmed by β-galactosidase staining. BMSCs with oxidative damage and senescence were treated with Jiangu Erxian pills containing active ingredients, and the effects of the drug-supplemented serum on metabolic activity of oxidatively aged BMSCs were detected by MTT assays at 24, 48, and 72 h. Apoptosis in senescent BMSCs was assessed by flow cytometry after 24 hours of drug intervention. Reverse transcription quantitative real-time PCR (RT-q real-time PCR) was used to observe changes in p16INK4 and p21CIPI and p53 expression levels related to senescence. Results: Treatment with 0.5 mmol/L H2O2 for 24 hours induced morphological changes of senescence in BMSCs, with a β-galactosidase positive staining rate exceeding 90%. Treatment with 5% concentration of Jiangu Erxian Pills containing active ingredients for 24 h and 48 h and 10% drug concentration for 24 h promoted cell proliferation (P<0.05). However, a 20% concentration inhibited proliferation after 48 hours (P<0.05). Treatment with 10% drug-containing serum for 24 hours significantly inhibited apoptosis (P<0.05) and downregulated the expression of p21CIP1 and p53 (P<0.05), while no statistically significant difference was observed in p16INK4 expression (P>0.05). Conclusion: Jiangu Erxian pills can promote the proliferation of oxidatively aged BMSCs, enhance the metabolism of rat BMSCs, markedly reduce their apoptosis rate, and achieve certain regulatory effects on the expression of aging-related genes p21CIPI and p53. The antioxidant and anti-aging effects are achieved through multiple pathways.
Key words：Jiangu Erxian pill; oxidative stress; senescence; bone marrow mesenchymal stem cells; proliferation; apoptosis
发布时间：2025-11-29
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