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

[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。

[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.