| 王家伟,孙廉旭,张 莎,王 文.黄芪甲苷激活TGF-?茁1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究[J].,2018,(8):1407-1413 |
| 黄芪甲苷激活TGF-?茁1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究 |
| Astragaloside induces Bone Marrow Mesenchymal Stem Cells Differentiating into Pericytes through TGF-β1/Smad2 Signaling Pathway |
| 投稿时间：2017-09-12 修订日期：2017-10-30 |
| DOI：10.13241/j.cnki.pmb.2018.08.002 |
| 中文关键词: 黄芪甲苷 周细胞 间充质干细胞 TGF-β1/Smad2信号传导通路 |
| 英文关键词: Astragaloside MSCs Pericytes TGF-β1/Smad2 signaling pathway |
| 基金项目:国家自然科学基金项目(81373845) |
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| 中文摘要: |
| 摘要 目的：观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用，揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间充质干细胞为研究对象，采用全骨髓培养法，从SD大鼠乳鼠（5d-7d）股骨提取原代细胞，体外传代纯化，P4-P5代用于实验。实验分阴性对照组，大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum, FBS)向周细胞分化的作用，揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间培养基培养，不给予药物干预；阳性对照组，给予转化生长因子β1(TGF-β1) 5 ng/mL干预3天；黄芪甲苷(Astragaloside,Ast) 组，给予黄芪甲苷4 μg/mL分别干预1 d、2 d、3 d、5 d、7 d；阻断剂组，TGF-β1 受体阻断剂 sb431542 预处理后，再加入黄芪甲苷4 μg/mL分别诱导1 d、2 d、3 d。实时定量PCR( RT-PCR) 检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白( alpha smooth muscle actin,α-SMA )信使核糖核酸(mRNA)表达；印迹法(Western blot) 检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果：经黄芪甲苷诱导刺激后，大鼠骨髓间充质干细胞在mRNA和蛋白水平上，NG2、α-SMA 的表达量均升高；Ast 3d组，与阴性对照组和阳性对照组相比，NG2、?-SMA蛋白和mRNA的表达量均显著升高(p<0.05)。黄芪甲苷刺激细胞后，TGF-β1/Smad2信号传导通路被激活，从第一天开始，p-Smad2蛋白表达量升高，第3天到达顶峰，随后降低；其中第三天，p-Smad2蛋白表达量显著高于Ast 0d组(p<0.01) 。加入阻断剂后，黄芪甲苷受到 sb431542影响，对细胞的作用减弱，NG2、α-SMA蛋白和mRNA表达量均下调；sb431542+Ast 3d组，与Ast 3d组相比，NG2、α-SMA 蛋白和mRNA的表达量显著降低(p<0.001)。受阻断剂的影响，黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱，前三天p-Smad2蛋白的表达量均降低，sb431542+Ast 3d组，与Ast 3d组相比，p-Smad2蛋白的表达量显著降低(p<0.001)。结论：黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用，其机制与激活TGF-β1/Smad2信号传导通路相关。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of astragaloside on the differentiation of bone marrow mesenchymal stem cells into pericytes. Methods: The MSCs were isolated from SD rats (5d-7d) bone marrow by whole bone marrow primary culture method, purified by amplification in vitro, and the fourth and fifth generation were used in this study. The purified cells were divided into three groups: negative control group, incubated in L-DMEM with 15% FBS without drug intervention; positive control group,cultured with TGF-β1 5 ng/mL for 72 hour; Ast group, cultured with 4 μg/mL Ast for 1 day, 2 day, 3 day, 5 day and 7 day; blocker group,inhibitor of the TGF-β1 typeⅠreceptor sb431542 being administered prior to Ast exposure,of which samples were collected at 1 day, 2 day, 3 day. Expression levels of NG2, α-SMA were examined by RT-PCR and Western blot. The expression levels of p-Smad2 and Smad2/3 were examined by Western blot. Results: The mRNA and protein expression level of NG2, α-SMA was higher in all Ast group. The mRNA and protein expression level of NG2, α-SMA was higher in Ast 3d group, compared with the negative control group and the positive control group(p<0.05). The TGF-β1/Smad signaling was activated after Ast intervention. The p-Smad2 protein level increased by day 1, reached a maximum at day 3, and then decreased. The protein expression level of p-Smad2 was higher in Ast 3d group,compared with the Ast 0d group (p<0.01). The effect of the astragaloside is blocked by the inhibitor of the TGF-β1 typeⅠsb431542 exposure, leading to the down-regulation of the mRNA and protein level of NG2, α-SMA. Compared with the Ast group at the same time point, the mRNA and protein expression level of NG2, α-SMA reduced significantly in the blocker group at day3(p<0.001). The signaling pathway was inhibited, as the p-Smad2 protein level decreased. Compared with the Ast 3d group, the protein expression level of p-Smad2 reduced significantly in the sb431542+Ast 3d group(p<0.001). Conclusion: Astragaloside can induce bone marrow mesenchymal stem cells differentiating into pericytes through the TGF-β1/Smad2 signaling pathway. |
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