多聚磷酸盐促进肌腱干细胞增殖和能量代谢的体外研究△

Polyphosphate promotes tendon stem cell proliferation and energy metabolism: a study in vitro

摘要:

［目的］ 探讨多聚磷酸盐 （inorganic polyphosphate, Poly P） 对体外肌腱干细胞增殖和能量代谢的影响。［方法］ 分离培养大鼠肌腱干细胞，采用流式细胞术对其表面分子进行鉴定，然后用 0、0.5、1.0 mmol/L 的 Poly P 处理大鼠肌腱干细胞，行 CKK-8、细胞周期实验和 ATP 能量检测，采用蛋白免疫印迹（Western blot）和免疫荧光检测 I 型胶原的表达。［结果］流式细胞分析表明>95%的分离细胞均表达 CD44 和 CD90.1，几乎不表达 CD45 和 CD106。 CKK-8 检测表明：随着培养时间的推移， 三个浓度 Poly P 组的 OD 值均显著增加 （P<0.05），培养 4~24 h，OD 值呈浓度依赖性增加，三组间差异均有统计学意义 （P< 0.05）。流式细胞检测表明，Poly P 呈浓度依赖性提高细胞 G2+S 期的百分比，差异有统计学意义（P<0.05）。ATP 检测方面，随培养时间的推移，三个浓度 Poly P 组的 ATP 检测的标准值均显著增加 （P<0.05）。Poly P 可以促进大鼠肌腱干细胞的 ATP 合成，且呈浓度依赖性，培养 4~24 h，三组间 ATP 检测的比值差异均有统计学意义（P<0.05）。免疫荧光染色方面，可见随 Poly P 的增加，I 型胶原的荧光显色强度显著增加。Western blot 检测表明 0.5、1.0 mmol/L 的 Poly P 组比较 0 mmol/L Poly P 组的 I 型胶原的表达显著增加，差异有统计学意义（P<0.05）。［结论］在体外 Poly P 可以上调大鼠肌腱干细胞基质合成基因 I 型胶原的表达和 ATP 的生成，促进肌腱干细胞的增殖。

Abstract:

[Objective] To investigate the effects of inorganic polyphosphate (Poly P) on the proliferation and energy metabolism of ten- don stem cells in vitro. [Methods] Rat tendon stem cells were isolated and cultured, their surface molecules were identified by flow cytome- try, and then treated with 0 mmol/L, 0.5 mmol/L, and 1.0 mmol/L Poly P. The Ckk-8, cell cycle and ATP energy assays were performed. In addition, the immunofluorescence and western blot assays were used to detect the expression of type I collagen. [Results] The flow cytome- try showed that ＞ 95% of the isolated cells expressed CD44 and CD90.1, but almost no CD45 and CD106. In term of Ckk-8 assay, the OD values of in all groups increased significantly over time (P＜0.05) , which increased significantly in Poly P concentration-dependent manner, with statistically significant differences among the three groups from 4 to 24 hours (P＜0.05) . The flow cytometry revealed that Poly P in- creased the percentage of G2+S phase and promoted cell proliferation in dosage-dependent manner with a statistically significant differenc- es (P＜0.05) . In terms of ATP detection, the ATP detection in the three Poly P groups increased significantly over time (P＜0.05) , which sig- nificantly increased in Poly P concentration-dependent manner with statistically significant differences among the three groups from 4 to 24 hours (P＜0.05) . Regarding to type I collagen presentation, the immunofluorescence staining showed that the fluorescence intensity of type I collagen increased significantly with the increase of Poly P concentrations, additionally, Western blot analysis showed that the expression of type I collagen in 0.5 mmol/L and 1.0 mmol/L Poly P group was significantly increased compared with that in 0 mmol/L Poly P group (P＜ 0.05) . [Conclusion] Poly P does promote the proliferation of rat tendon stem cells by up-regulating the expression of type I collagen and ATP production in vitro.