摘要
目的：探讨疏血通脉胶囊含药血清对大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）凋亡的影响以及作用机制。方法：体外培养大鼠BMSCs,以不同浓度双氧水（H2O2）诱导BMSCs凋亡,取最适宜浓度1.0 mmol·L^-1用于实验。疏血通脉胶囊低、中、高剂量（含生药1.81,3.62,7.24 g·kg^-1）灌胃给药制备大鼠含药血清。选择第3代BMSCs,随机分正常组（不做处理,加入体积分数为15%正常大鼠血清培养液）,模型组（H2O2诱导细胞凋亡,加入体积分数为15%正常大鼠血清培养液）和疏血通脉胶囊低、中、高剂量含药血清组（H2O2诱导细胞凋亡,分别加入对应剂量、体积分数为15%的疏血通脉胶囊含药血清培养液）,共5组,作用24 h后噻唑蓝比色法（MTT）检测细胞相对存活率,Annexin V-FITC/PI流式细胞术检测细胞凋亡率,实时荧光定量PCR法（Real-time PCR）检测半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）和B细胞淋巴瘤/白血病-2（Bcl-2）mRNA的表达。结果：H2O2能体外模拟缺血缺氧环境诱导BMSCs凋亡。MTT结果显示疏血通脉胶囊低、中、高剂量含药血清组细胞存活率较模型组有明显提高（P〈0.01）,且存活率随剂量增加而增高（P〈0.05）;Annexin V-FITC/PI流式细胞术结果显示疏血通脉胶囊含药血清可以降低H2O2诱导的BMSCs凋亡率（P〈0.01）。Real-time PCR结果显示H2O2诱导可以增加BMSCs的Caspase-3 mRNA表达,降低Bcl-2 mRNA表达。疏血通脉胶囊含药血清处理下调了Caspase-3 mRNA的表达（P〈0.01）,上调了Bcl-2 mRNA的表达（P〈0.01）。结论：疏血通脉胶囊含药血清可通过上调Bcl-2基因表达,负性调控Caspase-3基因,从而抑制H2O2导致的BMSCs凋亡。
Objective： To investigate the effect and mechanism of Shuxue Tongmai capsule-containing serum on apoptosis in rat bone marrow mesenchymal stem cells（BMSCs）. Method： BMSCs were cultured with different concentrations of H_2O_2 in vitro to induce apoptosis,and then 1. 0 mmol·L^-1 optimum concentration was used for the experiment. First,Shuxue Tongmai capsule-containing serum were prepared at low（1. 81 g·kg^-1）,middle（3. 62 g·kg^-1） and high（7. 24 g·kg^-1） doses. Then the third-generation BMSCs were divided into 5 groups in random. The first group was control group,which only added with normal rat serum with a volume fraction of 15% in culture medium. The second group was model group,which was added with normal rat serum with a volume fraction of 15% in culture medium and the apoptosis cells were induced with H_2O_2. The other three groups were drug-contained serum groups,which were respectively prepared at low（0. 32 g·kg^-1）,middle（0. 64 g·kg^-1） and high（1. 28 g·kg^-1） doses,and added with drug-contained serum with a volume fraction of15%,and the apoptosis cells were induced with H_2O_2 in each group. After 24 hours,the cells＇ relative survival rate was detected by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT） method,and the apoptosis rate was evaluated by Annexin V-FITC/PI flow cytometry method. And cysteine aspartate protease-3（Caspase-3） and B-cell lymphoma-2（Bcl-2） mRNA expressions were assayed by Real-time PCR（Real-time PCR）. Result： H_2O_2 could imitate the ischemic anoxic environment in vitro and induce the apoptosis of BMSCs.The MTT detection results showed that the cells＇ survival rates of Shuxue Tongmai capsule-containing serum groups prepared at low,medium and high doses were significantly higher than that in the model group（P〈0. 01）,and the survival rate increased with the dose concentration（P〈0. 05）. And the Annexin V-FITC/PI flow cytometry results showed that the drug-containing serum of Shuxue Tongmai capsule could decrease the apoptosis rates of BMSCs induced with H_2O_2（P〈0. 01）. The Real-time PCR results showed that the induction with H_2O_2 could increase BMSCs＇ Caspase-3 mRNA expression and reduce their Bcl-2 mRNA expression. The treatment of Shuxue Tongmai capsule-containing serum could decrease Caspase-3 mRNA expression（P〈0. 01）,and increase Bcl-2 mRNA expression（P〈0. 01）. Conclusion： Shuxue Tongmai capsule-containing serum can negatively regulate Caspase-3 gene by increasing Bcl-2 gene expression,so as to inhibit the apoptosis of BMSCs caused with H_2O_2.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2018年第3期154-159,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
广西自然科学基金项目(2014GXNSFBA118168)
作者简介
黄一挚，讲师，从事药学与实践研究，Tel：0771—2287060，E-mail：yizhi0771@163.com;[通信作者]陈朝，硕士，副主任药师，从事医院药学与中药制剂研究，Tel：0771-5645433，E-mail：chaochen1980@163．com
