补阳还五汤含药血清对骨髓间充质干细胞增殖及细胞周期的影响

Effect of Buyang Huanwutang Serum on Proliferation and Cycle of Bone Mesenchymal Stem Cells

目的:研究补阳还五汤含药血清对骨髓间充质干细胞(BMSCs)增殖及细胞周期的影响。方法:以6.5,13,26 g·kg^(-1)补阳还五汤和等量蒸馏水连续灌胃SD大鼠1周,制备3种补阳还五汤含药血清和空白血清。将BMSCs分为补阳还五汤6.5 g·kg^(-1)组(用含10%6.5 g·kg^(-1)生药制备的血清培养),补阳还五汤13 g·kg^(-1)组(用含10%13 g·kg^(-1)生药制备的血清培养),补阳还五汤26 g·kg^(-1)组(用含10%26 g·kg^(-1)生药制备的血清培养),空白组(用含10%空白血清培养)。采用噻唑蓝(MTT)比色法检测各组BMSCs的增殖活性,流式细胞术检测细胞周期,蛋白免疫印迹(Western blot)法检测磷脂酰肌醇-3羟基激酶(PI3K)和磷酸化丝(苏)氨酸蛋白酶(p-Akt)(Ser473)蛋白的表达。结果:13,26 g·kg^(-1)生药制备的补阳还五汤含药血清作用BMSCs 48,72 h后,细胞增殖活性较空白组明显升高(P<0.05);3种补阳还五汤含药血清作用48 h后,BMSCs在S期的细胞比率随着药物浓度的升高而升高,与空白组比较有统计学意义(P<0.05);13,26 g·kg^(-1)生药制备的补阳还五汤含药血清作用48 h后,PI3K和p-Akt(Ser473)蛋白表达较空白组显著增强(P<0.01)。结论:13,26 g·kg^(-1)生药制备的补阳还五汤含药血清可能通过活化PI3K/Akt信号通路促使细胞进入S期,增强BMSCs增殖活性。

Objective： To investigate the effect of Buyang Huanwutang serum on proliferation and cycle of bone mesenchymal stem cells （BMSCs）. Method： Buyang Huanwutang （6.5,13,26 g·kg^-1） and equivalent distilled water were administered （ig） to SD rats for a week, so as to prepare three kinds of Buyang Huanwutang serum and the blank serum. BMSCs cells were divided into Buyang Huanwutang 6.5 g·kg-1 group （cultured with 10% serum from Buyang Huanwutang 6.5 g·kg^-1）, Buyang Huanwutang 13 g·kg^-1 group （cultured with 10% serum from Buyang Huanwutang 13 g·kg^-1）, Buyang Huanwutang 26 g·kg^-1 group （cultured with 10% serum from Buyang Huanwutang 26 g·kg^-1）, and blank group （cultured with 10% serum from distilled water）. Cell proliferative activity, cell cycle and protein expressions of phosphatidylinositol 3-kinase （PI3K）, phosphorylation serine/threonine kinase （p-Akt） （Ser473） were detected by MTT, flow cytometry and western blot respectively. Result： BMSCs proliferation was increased in Buyang Huanwutang 13, 26 g·kg^-1 groups after being cultured for 48,72 h, compared with the blank group （P〈0.05）. After being cultured with the three kinds of Buyang Huanwutang serum for 48 h, S-phase cell rate in BMSCs was significantly higher than that in blank group （P〈0.05）. Protein expressions of PI3K, p-Akt（Ser473） were up-regulated in BMSCs treated with serum from Buyang Huanwutang 13,26 g·kg^-1 for 48 h, compared with blank group （P〈0.01）. Conclusion： Serum from Buyang Huanwutang 13,26 g·kg^-1 may promote BMSCs into the S phase, and enhance the proliferation of BMSCs by activating PI3K/Akt signaling pathway.