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

文题释义：归肾丸：归肾丸原方出自《景岳全书》,全方由熟地、杜仲、山药、枸杞子、菟丝子、茯苓、当归、酒萸肉8味药组成,有补肾填精、调理冲任的功效,常用于治疗妇科疾病。作者采用纯水煎煮的方法获取归肾丸,所制得的归肾丸冻干粉1 g含生药量约为5.2 g。原代骨髓间充质干细胞：原代细胞是指直接从机体取下细胞、组织和器官后立即进行培养,传代前保持原有细胞的基本性质,通常把第1代至第10代以内的细胞统称为原代细胞。该实验从3周龄雄性SD大鼠的双侧股骨骨髓获取原代骨髓间充质干细胞,经200目筛网过滤后,用全骨髓贴壁法纯化,经鉴定后选取纯度和增殖能力均较高的第3代细胞进行后续实验。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。ORCID: 0000-0003-0720-4797(刘燕)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

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

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.