Bushen Huoxue Recipe extract at the optimal concentration promotes the proliferation of bone marrow mesenchymal stem cells of Sprague-Dawley rats

doi:10.3969/j.issn.2095-4344.1585

Abstract

Abstract:

BACKGROUND: It is unclear what is the optimal concentration of Bushen Huoxue Recipe to promote the proliferation and differentiation of bone marrow mesenchymal stem cells.
OBJECTIVE: To investigate the optimal concentration and induction time of Bushen Huoxue Recipe extract to induce the proliferation and differentiation of bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were isolated and purified by adherent screening culture. Then, the bone marrow mesenchymal stem cells of 3rd generation were cultured with the extracts of Bushen Huoxue Recipe at different concentration (0, 10, 20, 40, 100, 120, 150, 200, 300, 400, 500 and 600 mg/L). The induction time was 1, 3, 5 and 7 days, respectively. The growth of bone marrow mesenchymal stem cells induced by Bushen Huoxue Recipe extracts was observed by fluorescent inverted microscope. The toxicity of different concentration solutions to bone marrow mesenchymal stem cells was detected by cell counting kit-8 for screening the optimal drug concentration and induction time.
RESULTS AND CONCLUSION: Compared with the 0 mg/L group, 100, 120 and 150 mg/L extracts from Bushen Huoxue Recipe significantly promoted the proliferation of bone marrow mesenchymal stem cells at the 5th and 7th days (P < 0.05). The 150 mg/L Bushen Huoxue Recipe extract showed the strongest proliferative effect (P < 0.01), while the proliferation rate decreased significantly, when the mass concentration of Bushen Huoxue Recipe extract was 150-200 mg/L. With the extension of the induction time, the proliferation of bone marrow mesenchymal stem cells showed no obvious changes after intervention with Bushen Huoxue Recipe at a concentration of more than 200 mg/L.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Drugs, Chinese Herbal, Cell Proliferation, Tissue Engineering

CLC Number:

Wei Rong, Wen Xin, Wang Li, Wei Zhiyong. Bushen Huoxue Recipe extract at the optimal concentration promotes the proliferation of bone marrow mesenchymal stem cells of Sprague-Dawley rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 691-696.

Figures/Tables 2

2.1 SD大鼠骨髓间充质干细胞形态学变化培养第3天，细胞有触角伸出，细胞形态多数呈多角形、棱形，少部分呈现条索状，细胞为成簇状生长，胞浆内分布呈颗粒状。细胞培养至第7天，原代细胞长满瓶底，见图1A-C。传代培养，细胞进入快速生长期，第3代细胞形态均一，呈网状、纤维状、漩涡状生长，排列有一定方向性，见图1D-F。 2.2 SD大鼠骨髓间充质干细胞流式细胞鉴定结果第3代骨髓间充质干细胞CD44、CD29为阳性表达，分别为96.3%，77.1%，而CD34、CD45为弱表达，分别为1.2%，2.5%，见图2。 2.3 补肾活血方提取物干预后细胞形态改变骨髓间充质干细胞经不同质量浓度补肾活血方提取物干预后第1天，低质量浓度干预组形态未见明显改变，>300 mg/L时细胞开始凋亡，可见死亡细胞。第3天，低质量浓度干预组细胞呈现缓慢增殖，并且发现质量浓度为150 mg/L干预组细胞生长旺盛，见图3A；质量浓度为200 mg/L干预组细胞出现死亡，但数量不多，见图3B；> 400 mg/L时细胞全部死亡。第7天，低质量浓度干预组细胞缓慢增殖，质量浓度为150 mg/L干预组细胞生长旺盛，见图3C；质量浓度为200 mg/L干预组细胞仍然有增殖，但死亡细胞数量也较多，见图3D。 2.4 不同质量浓度补肾活血方提取物对骨髓间充质干细胞增殖的影响以各组所测得的吸光度值的平均值为纵坐标，补肾活血方提取物溶液质量浓度为横坐标，绘制细胞吸光度值趋势图，骨髓间充质干细胞的吸光度值在不同补肾活血方提取物溶液质量浓度条件下的趋势，见图4。从图4可见，骨髓间充质干细胞加入不同质量浓度补肾活血方提取物进行干预，培养至1，3，5，7 d时加入CCK-8，所测得的吸光度值不同，随着补肾活血方提取物质量浓度的增加，测得的吸光度值逐渐增加，在药物质量浓度为150 mg/L时，测得吸光度值接近峰值，当药物质量浓度> 200 mg/L时，吸光度值出现下降趋势。补肾活血方提取物干预骨髓间充质干细胞第7天时的吸光度值普遍较高，干预第1天时的吸光度值普遍较低，吸光度值随着补肾活血方提取物溶液干预时间的增加出现上升趋势。由表1可见，与对照组(0 mg/L)比较，第5，7天时，100，120，150 mg/L补肾活血方提取物可促进骨髓间充质干细胞增殖(P < 0.05)；在第7天时，150 mg/L补肾活血方提取物促增殖作用最强(P < 0.01)；药物质量浓度在150-200 mg/L时，增殖率下降明显；随着实验时间的延长，> 200 mg/L时，补肾活血方提取物对骨髓间充质干细胞增殖作用不明显。"

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