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黄勇,董克芳,王凡,杨强健.三种补肾方含药血清对衰老骨髓间充质干细胞中COL Ⅰ和ALP的影响[J].湖南中医药大学学报,2022,42(7):1082-1086[点击复制] |
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| 三种补肾方含药血清对衰老骨髓间充质干细胞中COL Ⅰ和ALP的影响 |
| 黄勇,董克芳,王凡,杨强健 |
| (湖南中医药大学, 湖南 长沙 410208;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 研究健骨二仙丸、金匮肾气丸和六味地黄丸含药血清对衰老骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）成骨分化过程中Ⅰ型胶原蛋白（collagen type Ⅰ,COL Ⅰ）和碱性磷酸酶（alkaline phosphatase,ALP）表达的影响，探讨3种补肾方治疗骨质疏松症的机制。方法 将40只SD大鼠分为健骨二仙丸组、金匮肾气丸组、六味地黄丸组、空白对照组，每组10只。分别灌胃75.9 g/kg健骨二仙丸、89.1 g/kg金匮肾气丸、85.8 g/kg六味地黄丸和等体积生理盐水，每天定时灌胃2次，连续7 d，制备含药血清。用H2O2将P3代BMSCs处理成衰老细胞，采用β-半乳糖苷酶染色进行观察。衰老BMSCs分为6组：模型组、诱导组、健骨二仙丸含药血清组、金匮肾气丸含药血清组、六味地黄丸含药血清组、空白血清组，于诱导第4、8、12、16天采用Real-time PCR法和Western blot法检测COL Ⅰ mRNA及蛋白表达水平，并在第4、8天时收集细胞上清液检测ALP活性。结果 衰老BMSCs被β-半乳糖苷酶染色成深蓝色，而正常细胞颜色无明显改变。在第4、8、12、16天，与模型组比较，诱导组、空白血清组COL Ⅰ表达水平均升高（P＜0.05）；与诱导组、空白血清组比较，健骨二仙丸含药血清组、金匮肾气丸含药血清组COL Ⅰ表达水平均升高（P＜0.05）。在第4、12天，与诱导组相比较，六味地黄丸含药血清组COL Ⅰ表达水平升高（P＜0.05）。在第4、8天，与模型组比较，诱导组、空白血清组ALP活性表达均明显升高（P＜0.05）。与诱导组、空白血清组比较，健骨二仙丸含药血清组、金匮肾气丸含药血清组、六味地黄丸含药血清组ALP活性表达明显升高（P＜0.05）。结论 健骨二仙丸、金匮肾气丸和六味地黄丸均能增加衰老BMSCs成骨分化过程中COL Ⅰ的表达和ALP活性。 |
| 关键词: 健骨二仙丸 金匮肾气丸 六味地黄丸 衰老骨髓间充质干细胞 成骨分化 Ⅰ型胶原蛋白 碱性磷酸酶 |
| DOI：10.3969/j.issn.1674-070X.2022.07.005 |
| 投稿时间：2021-08-02 |
| 基金项目:湖南省自然科学基金——科卫联合项目（2018JJ6113）。 |
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| Effects of containing serum of three kinds of kidney tonifying prescriptions on COL Ⅰ and ALP in aging bone marrow mesenchymal stem cells |
| HUANG Yong,DONG Kefang,WANG Fan,YANG Qiangjian |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To study the effects of Jiangu Erxian Pill, Jinkui Shenqi Pill and Liuwei Dihuang Pill on the expression of collagen type Ⅰ (COL Ⅰ) and alkaline phosphatase (ALP) during the osteogenic differentiation of aging bone marrow mesenchymal stem cells (BMSCs), and to explore the mechanism of three kinds of kidney tonifying prescriptions in the treatment of osteoporosis. Methods Forty SD rats were divided into Jiangu Erxian Pill group, Jinkui Shenqi Pill group, Liuwei Dihuang Pill group and blank control group, with 10 rats in each group. They were given 75.9 g/kg Jiangu Erxian Pill, 89.1 g/kg Jinkui Shenqi Pill, 85.8 g/kg Liuwei Dihuang Pill and equal volume of normal saline by gavage twice a day for 7 d to prepare drug containing serum. P3 generation BMSCs were treated with H2O2 into senescent cells, β-galactosidase staining was used for observation. Aging BMSCs were divided into six groups:model group, induction group, Jiangu Erxian Pill containing serum group, Jinkui Shenqi Pill containing serum group, Liuwei Dihuang Pill containing serum group and blank serum group. On the 4th, 8th, 12th and 16th days of induction, the expression levels of COL Ⅰ mRNA and protein were detected by Real-time PCR and Western blot, and the cell supernatant was collected on the 4th and 8th days to detect ALP activity. Results Aging BMSCs were stained with β-galactosidase in dark blue, but the color of normal cells did not change significantly. On the 4th, 8th, 12th and 16th days, compared with the model group, the expression level of COL Ⅰ in the induction group and blank serum group increased (P<0.05). Compared with the induction group and blank serum group, the expression level of COL Ⅰ in Jiangu Erxian Pill containing serum group and Jinkui Shenqi Pill containing serum group increased (P<0.05). On the 4th and 12th days, compared with the induction group, the expression level of COL Ⅰ in Liuwei Dihuang Pill containing serum group increased (P<0.05). On the 4th and 8th days, compared with the model group, the expression of ALP activity in the induction group and blank serum group increased significantly (P<0.05). Compared with the induction group and blank serum group, the expression of ALP activity in Jiangu Erxian Pill containing serum group, Jinkui Shenqi Pill containing serum group and Liuwei Dihuang Pill containing serum group increased significantly (P<0.05). Conclusion Jiangu Erxian Pill, Jinkui Shenqi Pill and Liuwei Dihuang Pill can increase the expression of COL Ⅰ and ALP activity during osteogenic differentiation of aging BMSCs. |
| Key words: Jiangu Erxian Pill Jinkui Shenqi Pill Liuwei Dihuang Pill aging bone marrow mesenchymal stem cells osteogenic differentiation type I collagen alkaline phosphatase |
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