杜仲水提物上调Nur77表达促进骨髓间充质干细胞增殖和成骨分化  

Eucommia ulmoides Oliver aqueous extract promotes bone marrow mesenchymal stem cell proliferation and osteoblastic differentiation through upregulating Nur77 protein expression

作　　者：贺自克[1] 王上增[1] He Zike;Wang Shangzeng(Department of Osteoarthritis,Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450000,Henan Province)

机构地区：[1]河南省中医院骨关节科,河南省郑州市450000

出　　处：《中国组织工程研究》2023年第15期2371-2378,共8页Chinese Journal of Tissue Engineering Research

摘　　要：背景:骨髓间充质干细胞作为成骨细胞的前体细胞,探索其分化机制对于预防和治疗骨质疏松症具有重大意义。杜仲能够诱导骨髓间充质干细胞成骨分化,但是杜仲对骨髓间充质干细胞增殖、分化以及Nur77/MDM2/p53通路的影响尚不明确。目的:探究杜仲水提物对骨髓间充质干细胞增殖和分化的影响并探讨其作用机制。方法:杜仲水提物处理人源和鼠源骨髓间充质干细胞,采用CCK-8法和Annexin V-FITC/PI法分别检测细胞增殖和凋亡情况,Western blot检测增殖、凋亡相关蛋白的表达,Nur77及其下游MDM2、p53相关蛋白的表达及泛素化前后的蛋白水平。成脂、成骨定向诱导分化后,Western blot检测脂肪细胞和成骨细胞的标志蛋白表达来评估其分化程度。转染Nur77 siRNA进入10 mg/mL杜仲水提物处理的骨髓间充质干细胞,检测细胞增殖、凋亡和分化情况。最后,敲低Nur77之后,使用p53抑制剂Pifithrin-α处理骨髓间充质干细胞,检测细胞增殖、凋亡和分化情况以明确其作用机制。结果与结论:①杜仲水提物可促进2种骨髓间充质干细胞的增殖和成骨分化,抑制凋亡和成脂分化;②杜仲水提物可上调Nur77、MDM2蛋白表达,促进p53蛋白泛素化;敲低Nur77基因则抑制MDM2蛋白表达及p53蛋白泛素化,促进p53蛋白表达,抑制2种骨髓间充质干细胞增殖和成骨分化;③p53抑制剂可逆转Nur77 siRNA的作用,促进2种骨髓间充质干细胞的增殖和成骨分化,抑制凋亡和成脂分化;④结果表明,杜仲水提物可以通过Nur77/MDM2/p53途径促进2种骨髓间充质干细胞的增殖和成骨分化,为杜仲在骨相关疾病方面的研究提供理论参考。BACKGROUND:Bone marrow mesenchymal stem cells are precursor cells of osteoblasts.Exploring its differentiation mechanism is of great significance for guiding the prevention and treatment of osteoporosis.Eucommia ulmoides Oliver can induce the osteogenic differentiation of bone marrow mesenchymal stem cells.However,it is unclear about the effects of Eucommia ulmoides Oliver on proliferation and differentiation of bone marrow mesenchymal stem cells and Nur77/MDM2/p53 pathway.OBJECTIVE:To investigate the effect and mechanism of Eucommia ulmoides Oliver aqueous extract on bone marrow mesenchymal stem cell proliferation and differentiation.METHODS:The aqueous extract from Eucommia ulmoides Oliver bark was used to treat human and mouse bone marrow mesenchymal stem cells.The CCK-8 assay and Annexin V-FITC/PI assay were used to detect the proliferation and apoptosis of bone marrow mesenchymal stem cells.The expression of proliferation and apoptosis related proteins and the expression of Nur77 and its downstream MDM2,p53 pathway related proteins and the ubiquitination of p53 protein were detected by western blot assay.After directed induction of adipogenic and osteogenic differentiation,the degree of differentiation was evaluated by detecting the expression of adipocytes and osteoblasts marker proteins using western blot assay.Nur77 small interfering RNA(siRNA)was transfected into bone marrow mesenchymal stem cells treated with 10 mg/mL Eucommia ulmoides Oliver aqueous extract.Cell proliferation,apoptosis and differentiation were detected.Finally,after knocking down Nur77,bone marrow mesenchymal stem cells were treated with p53 inhibitor(Pifithrin-α),and the changes of cell proliferation,apoptosis and differentiation were detected to clarify the mechanism.RESULTS AND CONCLUSION:(1)The Eucommia ulmoides Oliver aqueous extract promoted the proliferation and osteoblastic differentiation of bone marrow mesenchymal stem cells,inhibited cell apoptosis and adipogenic differentiation.(2)Eucommia ulmoides Oliver aqueous extract u

关 键 词：骨髓间充质干细胞 杜仲 水提物 成骨分化 Nur77 MDM2 p53 通路 泛素化 

分 类 号：R394.2[医药卫生—医学遗传学] R318[医药卫生—基础医学] R-331