牛膝多糖通过Notch1通路影响骨髓间充质干细胞向髓核样细胞分化的机制  

Effect of achyranthes bidentata polysaccharides on the differentiation of bone mesenchymal stem cells into nucleus nulposus-like cells via Notch1 pathway

作　　者：岳宗进 刘汝银 于露 王新立 徐向阳 YUE Zongjin;LIU Ruyin;YU Lu;WANG Xinli;XU Xiangyang(Department of Spine,Henan Province Hospital of Traditional Chinese Medicine/The Second Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China)

机构地区：[1]河南省中医院/河南中医药大学第二附属医院脊柱科,河南郑州450000

出　　处：《西安交通大学学报（医学版）》2022年第4期600-606,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：河南省中医药科学研究专项基金项目(No.2019ZY2041)。

摘　　要：目的探讨牛膝多糖(achyranthes bidentata polysaccharides,ABPS)对骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向髓核样细胞分化的影响及机制。方法取5只4周龄SD大鼠,处死后解剖分离BMSCs,反复贴壁纯化,取第3代BMSCs,不同浓度ABPS作用48 h,MTT法检测细胞存活率,选择对BMSCs生长无毒性的ABPS最高浓度进行后续实验。根据处理方式不同将细胞分为对照组(常规培养)、ABPS组(200 mg/L ABPS)、Notch1组(转染Notch1)、Notch1阴性对照组(转染Notch1阴性对照)和ABPS+Notch1组(转染Notch1后加入200 mg/L ABPS)。诱导培养14 d后MTT法检测细胞增殖能力,qPCR和Western blotting分别检测collagenⅡ、聚集蛋白聚糖(aggrecan)、Notch1、Hes1 mRNA和蛋白表达水平,取第1、4、7、10、14天细胞培养基阿尔新蓝法检测葡糖胺聚糖(GAG)。结果400 mg/L ABPS作用BMSCs 48 h,细胞存活率与对照组比较显著降低(P<0.05),浓度≤200 mg/L时,ABPS对BMSCs生长无明显毒性作用,选择200 mg/L ABPS进行后续实验。诱导培养14 d后,与对照组比较,ABPS组细胞A值、培养基内GAG含量、collagenⅡ、aggrecan mRNA和蛋白相对表达量升高,Notch1、Hes1 mRNA和蛋白相对表达量降低;而Notch1组细胞A值、培养基内GAG含量、collagenⅡ、aggrecan mRNA和蛋白相对表达量降低,Notch1、Hes1 mRNA和蛋白相对表达量升高(P<0.05);ABPS+Notch1组可部分逆转过表达Notch1对BMSCs向髓核样细胞分化的抑制作用。结论ABPS可以促进BMSCs向髓核样细胞分化,作用机制可能与抑制Notch1通路有关。Objective To investigate the effect and mechanism of achyranthes bidentata polysaccharides(ABPS)on the differentiation of bone mesenchymal stem cells(BMSCs)into nucleus pulposus-like cells.Methods Five 4-week-old SD rats were sacrificed and their BMSCs were isolated and purified by repeated adherent method.The third-generation BMSCs were treated with different concentrations of ABPS for 48 h.The cell viability was detected by MTT method.The highest concentration of ABPS was selected for subsequent experiments.According to different treatment methods,cells were divided into control group(conventional culture),ABPS group(200 mg/L of ABPS),Notch1 group(transfected with Notch1),Notch1 negative control group(transfected with Notch1 negative control),and ABPS+Notch1 group(200 mg/L of ABPS was added after transfection with Notch1).After 14 days of induction,the cell survival rate was measured by MTT method.CollagenⅡwas detected by immunohistochemical staining.The mRNA and protein expression levels of collagenⅡ,aggrecan,Notch1 and Hes1 were detected by qPCR and Western Blotting respectively.The glucosaminoglycan(GAG)was detected by alcian blue method on the 1st,4th,7th,10th and 14th day of cell culture.Results After BMSCs were treated with 400 mg/L of ABPS for 48 h,the cell survival rate was significantly lower than that in the control group(P<0.05).When the concentration was≤200 mg/L,ABPS had no significant toxic effect on the growth of BMSCs.After 14 days of induction,compared with the control group,A value,GAG content in the medium,the expressions of collagen II and aggrecan mRNA and protein in the ABPS group were increased,and the expressions of Notch1 and Hes1 mRNA and protein were decreased(P<0.05).The relative cell viability,GAG content in the medium,the expressions of collagen II and aggrecan mRNA and protein in the Notch1 group were decreased,and the expressions of Notch1 and Hes1 mRNA and protein were increased(P<0.05).ABPS+Notch1 could partially reverse the inhibitory effect of Notch1 overexpression on t

关 键 词：骨髓间充质干细胞 髓核样细胞 细胞分化 牛膝多糖 Notch1通路 

分 类 号：R285.[医药卫生—中药学]