归肾丸水提物干预SD大鼠骨髓间充质干细胞增殖及PI3K、AKT蛋白的表达  

Guishen Pill water extract intervenes with the proliferation of bone marrow mesenchymal stem cells and expression levels of PI3K and AKT in Sprague-Dawley rats

作　　者：刘燕[1] 关永格 宋阳[1] Liu Yan;Guan Yongge;Song Yang(Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;the Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510360,Guangdong Province,China)

机构地区：[1]广州中医药大学,广东省广州市510405 [2]广州中医药大学第三附属医院,广东省广州市510360

出　　处：《中国组织工程研究》2020年第13期1983-1988,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81303000)，项目负责人：宋阳;广东省科技计划项目(2017A020215119)，项目负责人：关永格;广东省普通高校特色创新项目(2017KTSCX041)，项目负责人：宋阳~~

摘　　要：背景:补肾类方剂对骨髓间充质干细胞的促增殖作用已经被证实,但其作用机制尚不明确,该研究以归肾丸为例,探讨其可能的作用机制。目的:通过观察归肾丸水提物对骨髓间充质干细胞增殖的影响,探究归肾丸滋补肾精与骨髓间充质干细胞增殖之间的关系。方法:从3周龄雄性SD大鼠股骨骨髓中分离提取骨髓间充质干细胞,采用全骨髓贴壁法将其纯化,选用纯度较高的第3代细胞进行药物干预,将其分为空白对照组和10,20,40,80,100 mg/L归肾丸水提物组,在体外连续培养5 d以观察量效关系和时效关系。倒置显微镜下观察细胞形态,CCK-8法评估细胞增殖活性,Western blot检测细胞中PI3K、AKT蛋白表达。结果与结论:①归肾丸水提物组的细胞增殖能力明显高于空白对照组,存在一定的量效和时效关系,尤以80 mg/L归肾丸水提物干预3 d时作用效果最佳;②归肾丸水提物组骨髓间充质干细胞中PI3K、AKT蛋白的表达明显高于空白对照组;③结果提示,归肾丸水提物能有效促进骨髓间充质干细胞的增殖,可能与其参与调控PI3K/AKT信号通路有关。BACKGROUND:The effect of kidney-tonifying prescriptions on proliferation of bone marrow mesenchymal stem cells has been verified,but its mechanism of action is still unclear.In this study,we took Guishen Pil as an example to explore its functional mechanism.OBJECTIVE:To explore the relationship between Guishen Pill nourishing kidney essence and proliferation of bone marrow mesenchymal stem cells by observing the effect of Guishen Pill water extract on the proliferation of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated from the femoral bone marrow of 3-week-old male Sprague-Dawley rats and cultured in vitro.The cells were purified using the whole bone marrow adherence method,and the passage 3 cells of higher purity were selected for the drug intervention.The cells were divided into blank control group and 10,20,40,80 and 100 mg/L Guishen Pill groups.The dose-effect and time-effect relationships were investigated after cultured for 5 days.The cell morphology was observed under inverted microscope.The cell proliferation was evaluated by cell counting kit-8 assay.The expression levels of PI3 K and AKT proteins were detected by western blot assay.RESULTS AND CONCLUSION:Guishen Pill significantly increased the cell proliferation in the Guishen Pill groups in a dose-and time-effect manner.The proliferation of bone marrow mesenchymal stem cells in the 80 mg/L Guishen Pill group was strongest at the 3 rd day.The expression levels of PI3 K and AKT proteins in the Guishen Pill groups were significantly higher than those in the blank control group.In summary,Guishen Pill water extract can effectively boost the proliferation of bone marrow mesenchymal stem cells,which may be related to its involvement in regulating PI3 K/AKT signaling pathway.

关 键 词：骨髓间充质干细胞 归肾丸 细胞增殖 PI3K AKT 滋补肾精 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]