不同浓度葡萄糖诱导皮肤成纤维细胞老化的实验观察

Senescent effect of different concentrations of glucose on fibroblast

  • 摘要:
      背景  衰老是生理或病理过程综合作用的必然结果。研究如何延缓衰老,尤其是病理性衰老,可为许多疾病的治疗提供新的思路。而建立老化细胞模型正是衰老相关研究的基石。
      目的  探究含不同浓度葡萄糖的DMEM培养液诱导皮肤成纤维细胞老化的效果,为衰老研究提供实验基础。
      方法  分别采用含5.5 mmol/L、15 mmol/L、25 mmol/L、35 mmol/L和45 mmol/L葡萄糖的DMEM培养基培养成纤维细胞,通过CCK-8实验和细胞划痕试验观察细胞增殖、迁移功能的变化;对细胞进行β-半乳糖苷酶染色,判断细胞是否发生衰老;采用PCR检测衰老相关p53/p21通路中关键效应分子p53、p21和p16在不同组中的表达水平。
      结果  相较于5.5 mmol/L组,15 mmol/L组和25 mmol/L组成纤维细胞增殖和迁移功能提高,而35 mmol/L组和45 mmol/L组细胞增殖和迁移能力显著下降;β-半乳糖苷酶染色阳性的细胞比例随葡萄糖浓度的升高而增加;同时,p53、p21和p16的表达与葡萄糖浓度呈正相关。
      结论  高浓度葡萄糖(≥35 mmol/L)可抑制细胞的生物学功能,诱导细胞衰老,其分子机制可能是通过上调p53/p21通路引起p16表达水平的增高。

     

    Abstract:
      Background  Aging is the inevitable result of the comprehensive action of physiological or pathological processes. Research on how to delays aging, especially pathological aging, can provide new ideas for the treatment of many diseases. The establishment of aging cell model is the cornerstone of aging related research.
      Objective   To explore the effect of Dulbecco's Modified Eagle Medium (DMEM) with different concentrations of glucose on skin fibroblast aging.
      Methods   Fibroblasts were cultured in DMEM medium containing different concentrations of glucose (5.5 mmol/L, 15 mmol/L, 25 mmol/L, 35 mmol/L and 45 mmol/L). Cell proliferation and migration were detected by CCK-8 test and cell scratch assay, respectively. β-galactosidase staining was employed to determine the percentage of senescent cell. PCR was used to detect the expression levels of p53, p21 and p16 in each group to elucidate the underlying mechanism.
      Results   Compared with the 5.5 mmol/L group, the proliferation and migration of fibroblasts in the 15 mmol/L and the 25 mmol/L groups increased, while significantly decreased in the 35 mmol/L and the 45 mmol/L groups; the proportion of SA-β-gal positive cells increased along with the glucose concentration. Meanwhile, the expressions of p53, p21 and p16 were also positively correlated with glucose concentrations.
      Conclusion  Excessively high concentration of glucose (≥35 mmol/L) inhibits the biological function of cells and induces cell senescence. The pathogenesis is associated with increased expression of p16 via upregulation of p53/ p21 pathway.

     

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