20(S)-原人参二醇对大鼠骨髓间充质干细胞成骨分化的影响及其机制  

Effect of 20(S)-protopanaxadiol on osteogensis differentiation of bone marrow mesenchymal stem cells in rats and its mechanism

作　　者：张好 王翠竹[2] 皇甫慧敏 张新威 张一迪 周延民[1] ZHANG Hao;WANG Cuizhu;HUANGFU Huimin;ZHANG Xinwei;ZHANG Yidi;ZHOU Yanmin(Department of Plantation,Stomatology Hospital,Jilin University,Changchun 130021,China;Department of Pharmaceutical Design,School of Pharmaceutical Sciences,Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学口腔医院种植科,吉林长春130021 [2]吉林大学药学院药物设计学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2023年第4期867-874,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(82071152)。

摘　　要：目的:探讨20(S)-原人参二醇(PPD)对大鼠骨髓间充质干细胞(BMSCs)成骨分化的作用,并阐明其成骨诱导机制。方法:采用全骨髓法提取SD大鼠BMSCs,分为对照组和不同剂量(2.5、5.0、10.0、20.0和40.0 mg·L^(-1)) PPD组,采用CCK-8法检测各组BMSCs增殖活性,采用Calcein/PI染色法观察各组BMSCs存活情况,筛选合适浓度PPD用于后续实验。将BMSCs分为对照组和PPD组,于成骨诱导第7天,采用BCIP/NBT比色法进行碱性磷酸酶(ALP)染色并定量检测各组BMSCs中ALP活性;成骨诱导第21天,采用茜素红染色检测各组BMSCs中矿化结节形成情况并定量分析细胞矿化活性;成骨诱导第7天,采用实时荧光定量PCR (RT-qPCR)法检测各组BMSCs中ALP、1型胶原蛋白(COL^(-1))、骨钙素(OCN)和Runt相关转录因子2 (Runx-2) mRNA表达水平,免疫荧光染色法检测各组BMSCs中Runx-2和OCN蛋白表达荧光强度。结果:PPD处理第1和3天,与对照组比较,40.0 mg·L^(-1) PPD组BMSCs增殖活性明显降低(P<0.01);PPD处理第7天,与对照组比较,2.5、 5.0和10.0 mg·L^(-1) PPD组BMSCs增殖活性差异无统计学意义(P>0.05),20.0和40.0 mg·L^(-1) PPD组BMSCs增殖活性明显降低(P<0.01)。培养第1和3天,5.0、10.0和20.0mg·L^(-1) PPD组活细胞数较多,故选用10.0 mg·L^(-1) PPD用于后续实验。成骨诱导第7天,与对照组比较,PPD组BMSCs的ALP活性明显升高(P<0.01);成骨诱导第21天,与对照组比较,PPD组BMSCs中矿化结节数明显增多,矿化活性明显升高(P<0.01);成骨诱导第7天,与对照组比较,PPD组BMSCs中ALP、 COL^(-1)、 OCN和Runx-2 mRNA表达水平明显升高(P<0.05),Runx-2和OCN蛋白表达荧光强度均明显升高(P<0.01)。结论:PPD可通过增加BMSCs ALP活性和钙盐沉积,上调ALP、COL^(-1)、OCN和Runx-2等成骨基因的表达进而促进BMSCs的成骨分化,PPD是一种有效的成骨诱导活性因子。Objective:To discuss the effect of 20(S)-protopanaxadiol(PPD)on the osteogensis differentiation of bone marrow mesenchymal stem cells(BMSCs)of the rats,and to clarify the osteogenesis induction mechanism.Methods:The BMSCs were extracted from the SD rats by whole bone marrow method,and the BMSCs were divided into control group and 2.5,5.0,10.0,20.0,and 40.0 mg·L^(-1) PPD groups;CCK-8 assay was used to detect the proliferation activities of BMSCs in various groups;the survival of BMSCs in various groups was observed by Calcein/PI staining;the suitable concentration of PPD for the subsequent experiments was selected.The BMSCs were divided into control group and PPD group.On the 7th day of osteogenesis induction,BCIP/NBT colorimetry was used to carry out alkaline phosphatase(ALP)staining and quantitatively detect the activities of ALP in the BMSCs in various groups;On the 21th day of osteogenesis induction,the formations of calcium nodules in the BMSCs in various groups were detected by Alizarin red staining and the mineralization activities of cells in various groups were quantitatively detected;On the 7th day of osteogenesis induction,real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of ALP runt-related transcription factor 2(Runx-2),typeⅠcollagen(COL-1),and osteocalcin(OCN)mRNA in cells in various groups;the immunofluorescence intensities of Runx-2 and OCN protein expressions in the BMSCs in various groups were measured by immunofluorescence staining.Results:On the 1st and 3rd days of PPD treatment,compared with control group,the proliferation activity of the BMSCs in 40.0 mg·L^(-1) PPD group was significantly decreased(P<0.01);on the 7th day of PPD treatment,compared with control group,there were no significant differences in the proliferation activities of the cells between 2.5,5.0,and 10.0 mg·L^(-1) PPD groups(P>0.05),the proliferation activities of the BMSCs in 20.0 and 40.0 mg·L^(-1) PPD groups were significantly decreased(P<0.01).On the 1st and 3rd days of

关 键 词：20(S)-原人参二醇 成骨 骨髓间充质干细胞 细胞分化 碱性磷酸酶 免疫荧光染色 

分 类 号：R285.5[医药卫生—中药学] Q2-33[医药卫生—中医学]