南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (12): 1784-1792.doi: 10.12122/j.issn.1673-4254.2020.12.13

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Palbociclib可诱导人肾小管上皮细胞周期阻滞及衰老

黄柳维，沈燕婷，刘崇斌，李彩珍，王 骏   

  • 出版日期:2020-12-20 发布日期:2020-12-28

Palbociclib induces cell cycle arrest and senescence of human renal tubular epithelial cells in vitro#br#

  • Online:2020-12-20 Published:2020-12-28

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摘要： 目的 探讨Palbociclib药物对肾小管上皮细胞周期进展及细胞增殖的影响。方法 体外培养肾小管上皮细胞HK-2分别给予不同浓度（0、1、5、10、20 μmol/L）的Palbociclib进行处理，通过细胞计数和CCK8法检测不同浓度及时间下Palbociclib对HK-2细胞增殖及细胞活力的影响。EDU染色检测不同浓度下Palbiciclib对HK-2细胞作用5 d后的细胞增殖情况。流式分析检测Palbociclib对HK-2细胞生长周期分布的影响。采用SA-β-Gal、C12FDG等衰老染色技术检测不同浓度下Palbociclib作用于HK-2细胞5 d后其对衰老表型的诱导情况。RT-PCR检测P16、P21、P53及衰老相关分泌表型的mRNA相对表达水平，Western blot检测P16、P21和P53的蛋白表达情况。结果 Palbociclib抑制HK-2细胞增殖并诱导细胞周期阻滞于G1期。相比于对照组而言，高剂量（10 μmol/L）的 Palbociclib 可明显抑制 HK-2 细胞增殖活性，且在第 5 天时增殖抑制效果最为明显（P<0.01）。Palbociclib诱导HK-2细胞生长周期主要阻滞于G1期，而进入S期的细胞数相比于对照组来说明显减少（P<0.01）。Palbociclib可诱导HK-2细胞发生衰老，通过SA-β-Gal和C12FDG衰老染色检测，结果表明在Palbociclib作用下，HK-2细胞的胞内衰老相关半乳糖苷酶活性明显升高。RT-PCR及Western blot结果显示Palbociclib可明显上调HK-2细胞中P16、P21、P53等衰老相关因子的基因和蛋白表达水平（P<0.01）。与对照组相比，RT-PCR检测Palbociclib作用下的HK-2细胞中衰老相关分泌因子的基因表达水平也升高（P<0.01）。结论 Palbociclib可通过抑制HK-2细胞生长并阻滞其细胞周期进程进而诱导HK-2细胞发生衰老。

关键词: Palbociclib；肾小管上皮细胞；细胞增殖；细胞衰老

Abstract: Objective To investigate the effect of palbociclib on cell cycle progression and proliferation of human renal tubular epithelial cells. Methods Human renal tubular epithelial cell line HK-2 was treated with 1, 5, 10, and 20 μmol/L of palbociclib, and the changes in cell proliferation and viability were examined by cell counting and CCK8 assay. EDU staining was used to assess the proliferation of HK-2 cells following palbiciclib treatment at different concentrations for 5 days. The effect of palbociclib on cell cycle distribution of HK-2 cells was evaluated using flow cytometry. SA-β-Gal staining and C12FDG senescence staining were used to detect senescence phenotypes of HK-2 cells after palbociclib treatment at different concentrations for 5 days. The relative mRNA expression levels of P16, P21, and P53 and the genes associated with senescence-related secretion phenotypes were detected by RT-PCR, and the protein expressions of P16, P21 and P53 were detected by Western blotting. Results Palbociclib inhibited HK-2 cell proliferation and induced cell cycle arrest in G1 phase. Compared with the control cells, HK-2 cells treated with high-dose (10 μmol/L) palbociclib exhibited significantly suppressed cell proliferation activity, and the inhibitory effect was the most obvious on day 5 (P<0.01). Palbociclib treatment significantly reduced the number of cells in S phase (P<0.01) and induced senescence of HK-2 cells. The results of SA-β-Gal and C12FDG senescence staining showed a significantly enhanced activity of intracellular senescence-related galactosidase in
palbociclib-treated HK-2 cells, suggesting significant senescence of the cells (P<0.01). RT-PCR and Western blotting showed that palbociclib treatment significantly increased the mRNA and protein expression levels of P16, P21, and P53 in HK-2 cells (P<0.01); the mRNA expression levels of senescence-related secretory factors also increased significantly in HK-2 cells after palbociclib treatment (P<0.01). Conclusions Palbociclib induces HK-2 cell senescence by causing cell growth arrest and delaying cell cycle progression.

Key words: palbociclib; renal tubule epithelial cells; cell proliferation; cellular senescence