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杨强健,董克芳,王凡,林少如,黄勇,李为.三种补肾方含药血清对衰老骨髓间充质干细胞成骨分化及Runx2表达的影响[J].湖南中医药大学学报英文版,2023,43(1):53-58.[Click to copy
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| 三种补肾方含药血清对衰老骨髓间充质干细胞成骨分化及Runx2表达的影响 |
| 杨强健,董克芳,王凡,林少如,黄勇,李为 |
| (湖南中医药大学, 湖南 长沙 410208;湖南中医药大学第二附属医院, 湖南 长沙 410005) |
| 摘要: |
| 目的 探究3种补肾方（健骨二仙丸、六味地黄丸、金匮肾气丸）含药血清对衰老骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）成骨分化及Runt相关转录因子2（Runt-related transcription factor2,Runx2）表达的影响,研究3种补肾方治疗骨质疏松症的机制。方法 将40只SPF级SD大鼠随机分为空白对照组（等体积生理盐水）、健骨二仙丸组（75.9g/kg）、六味地黄丸组（85.8g/kg）及金匮肾气丸组（89.1g/kg），每组10只，分别灌胃用于制备含药血清。建立对数增长的P3代BMSCs。500μmol/L 30%H2O2处理以建立氧化衰老BMSCs模型。cck-8法检测衰老BMSCs代谢活性。衰老BMSCs分为模型组、诱导组、空白血清组、健骨二仙丸含药血清组、六味地黄丸含药血清组、金匮肾气丸含药血清组，共6组。在诱导第4、8、12、16天，观察各组细胞形态学变化，Western blot法检测Runx2蛋白表达，RT-PCR法检测Runx2 mRNA相对表达量。结果 与正常BMSCs比较，H2O2处理后的BMSCs活性显著降低（P<0.05）。对比模型组，诱导组和空白血清组衰老BMSCs在分化过程中，茜素红染色增多；对比诱导组及空白血清组，各含药血清组衰老BMSCs在分化过程中，茜素红染色增多；且除模型组外，各组随着时间推移，茜素红染色不断增加。在第4、8、12、16天，对比模型组，诱导组、空白血清组Runx2 mRNA和蛋白表达水平均升高（P<0.05）；对比诱导组、空白血清组，健骨二仙丸含药血清组、金匮肾气丸含药血清组Runx2 mRNA和蛋白表达水平均升高（P<0.05）。在第4、8、12天，对比诱导组，六味地黄丸含药血清组Runx2 mRNA和蛋白表达水平升高（P<0.05）。在第4、12、16天，对比空白血清组，六味地黄丸含药血清组Runx2 mRNA和蛋白表达水平升高（P<0.05）。结论 健骨二仙丸、六味地黄丸、金匮肾气丸均可促进Runx2的表达，并促进衰老BMSCs往成骨方向分化。 |
| 关键词: 健骨二仙丸 六味地黄丸 金匮肾气丸 衰老骨髓间充质干细胞 成骨分化 Runt相关转录因子2 |
| DOI：10.3969/j.issn.1674-070X.2023.01.009 |
| Received:June 24, 2022 |
| 基金项目:湖南省自然科学基金——科卫联合项目（2018JJ6113）；田心义名中医工作室。 |
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| Effects of serum containing three kidney-tonifying formulas on osteogenic differentiation and Runx2 expression of aging bone marrow mesenchymal stem cells |
| YANG Qiangjian,DONG Kefang,WANG Fan,LIN Shaoru,HUANG Yong,LI Wei |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The Second Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To investigate the effects of serum containing three kidney-tonifying formulas (Jiangu Erxian Pill, Liuwei Dihuang Pill, Jingui Shenqi Pill) on osteoblast differentiation and the expression of Runt-related transcription factor2 (Runx2) of aging bone marrow mesenchymal stem cells (BMSCs) and to study the mechanism of these three formulas in the treatment of osteoporosis. Methods A total of 40 SPF SD rats were randomly divided into blank control group (equal volume normal saline), Jiangu Erxian Pill group (75.9 g/kg), Liuwei Dihuang Pill group (85.8 g/kg) and Jingui Shenqi Pill group (89.1 g/kg),with 10 rats in each group. All the groups received intragastric administration of the corresponding materials respectively to prepare drug-containing serum. The P3 generation BMSCs with logarithmic growth were prepared and were further treated with 500 μmol/L 30% H2O2 to establish the model of oxidative aging BMSCs. The metabolic activity of aging BMSCs was detected by cck-8 method. The aging BMSCs were divided into 6 groups: model group, induction group, blank serum group, serum containing Jiangu Erxian Pill drug group, serum containing Liuwei Dihuang Pill group and serum containing Jingui Shenqi Pill group. On the 4th, 8th, 12th and 16th days of induction, the morphological changes of cells in each group were observed. The expressions of Runx2 protein were detected by Western blot and the relative expressions of Runx2 mRNA were detected by RT-PCR. Results Compared with normal BMSCs, the activity of BMSCs treated with H2O2 was significantly lower (P<0.05). Compared with model group, alizarin red staining increased during the differentiation of aging BMSCs in induction group and blank serum group. Compared with induction group and blank serum group, alizarin red staining increased during the differentiation of aging BMSCs in drug-containing serum groups. Alizarin red staining increased with time in all groups except model group. On the 4th, 8th, 12th and 16th days, Runx2 mRNA and protein expression levels increased in induction group and blank serum group in comparison to model group (P<0.05). Compared with induction group and blank serum group, mRNA Runx2 and protein expression levels in serum containing Jiangu Erxian Pill group and serum containing Jingui Shenqi Pill group increased (P<0.05). On the 4th, 8th and 12th days Runx2 mRNA and protein expression levels in serum containing Liuwei Dihuang Pill group were higher (P<0.05) than those in induction group. On the 4th, 12th and 16th days, Runx2 mRNA and protein expression levels in serum containing Liuwei Dihuang Pill group were higher (P<0.05) than those of the blank serum group. Conclusion Jiangu Erxian Pill, Liuwei Dihuang Pill and Jingui Shenqi Pill can all promote the expression of Runx2 and the osteogenic differentiation of aging BMSCs. |
| Key words: Jiangu Erxian Pill Liuwei Dihuang Pill Jingui Shenqi Pill aging bone marrow mesenchymal stem cells osteogenic differentiation Runt-associated transcription factor 2 |
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