抑瘤素M对人脐带间充质干细胞增殖和成骨分化的影响  

Effect of oncostatin M on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells

作　　者：孔宁[1] 周余来[1] 张璐 石毅[3] 石艳[4] 孙忠平 邹颖刚[5] KONG Ning;ZHOU Yulai;ZHANG LU;SHI Yi;SHI Yan;SUN Zhongping;ZOU Yinggang(Department of Biomedical Materials,School of Pharmacy,Jilin University,Changchun 130021,China;Jilin Zhong Ke Bio-engineering Co.,Ltd.,Changchun 130012,China;Center of Biological Engineering Experiment,School of Pharmacy,Jilin University,Changchun 130021,China;Department of Experimental Pharmacology and Toxicology,School of Pharmacy,Jilin University,Changchun 130021,China;Reproductive Center,Department of Obstetrics and Gynecology,Second Hospital,Jilin University,Changchun 130041,China)

机构地区：[1]吉林大学药学院医用生物材料学教研室,吉林长春130021 [2]吉林省中科生物工程股份有限公司,吉林长春130012 [3]吉林大学药学院生物工程实验中心,吉林长春130021 [4]吉林大学药学院实验药理与毒理学教研室,吉林长春130021 [5]吉林大学第二医院妇产科生殖中心,吉林长春130041

出　　处：《吉林大学学报（医学版）》2020年第4期699-706,I0001,共9页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅重点科技攻关项目资助课题(20170204036YY);吉林省科技厅自然科学基金项目资助课题(20180101140JC)。

摘　　要：目的:探讨抑瘤素M(OSM)对体外培养的人脐带间充质干细胞(hUCMSCs)增殖及成骨分化的影响,阐明OSM是一种有效的成骨诱导活性因子。方法:体外培养hUCMSCs,流式细胞术检测第3代hUCMSCs表面标志物;CCK-8法测定不同剂量(0.1、1.0和10.0μg·L^-1)重组人抑瘤素M(rhOSM)处理后hUCMSCs的增殖活性。将hUCMSCs分为对照组、经典成骨诱导剂组和不同剂量(0.1、1.0和10.0μg·L^-1)rhOSM组,于成骨诱导第4、7和14天,采用BCIP/NBT法进行碱性磷酸酶(ALP)染色并定量检测细胞中ALP活性,茜素红染色法检测钙结节的生成情况并半定量分析细胞矿化活性,实时荧光定量PCR(RT-qPCR)法检测hUCMSCs中Runt相关转录因子2(Runx2)和OCN mRNA表达水平。结果:hUCMSCs符合间充质干细胞(MSCs)鉴定标准。rhOSM处理hUCMSCs 72 h,与对照组比较,10.0μg·L^-1 rhOSM组细胞增殖活性明显降低(P<0.05);处理96 h,与对照组比较,各剂量rhOSM组细胞增殖活性均明显降低(P<0.05),且各剂量rhOSM组组间比较差异有统计学意义(P<0.01)。成骨诱导第4、7和14天,与经典成骨诱导剂组比较,各剂量rhOSM组细胞中ALP活性和Runx2 mRNA表达水平均明显升高(P<0.05);与0.1μg·L^-1 rhOSM组比较,1.0和10.0μg·L^-1 rhOSM组ALP活性和Runx2 mRNA表达水平明显升高(P<0.05);与成骨诱导第7天比较,诱导第14天各剂量rhOSM组细胞中ALP活性和Runx2 mRNA表达水平略降低,但差异无统计学意义(P>0.05)。成骨诱导第7、14和21天,与经典成骨诱导剂组比较,各剂量rhOSM组细胞矿化活性均明显升高(P<0.01);与0.1μg·L^-1 rhOSM组比较,1.0和10.0μg·L^-1 rhOSM组细胞矿化活性明显升高(P<0.05)。与经典成骨诱导组比较,各剂量rhOSM组OCN mRNA表达水平均明显升高(P<0.05);成骨诱导第7、14和21天,与0.1μg·L^-1 rhOSM组比较,1.0和10.0μg·L^-1 rhOSM组细胞中OCN mRNA表达水平明显升高(P<0.05),具有时间-剂量依赖性。结论:rhOSM通过上调Runx2和OCN的表达及增加成骨细胞AObjective:To investigate the effect of oncostatin M(OSM)on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCMSCs),and to elucidate that OSM is an effective osteogenic induction active factor.Methods:The hUCMSCs were cultivated in vitro.The surface antigens of hUCMSCs at the third generation were detected by flow cytometry.CCK-8 assay was used to determine the proliferation activities of hUCMSCs after treated by 0.1,1.0 and 10.0μg·L^-1 recombined human oncostatin M(rhOSM).The hUCMSCs were divided into control group,classical osteogenic induction group,and different doses(0.1,1.0 and 10.0μg·L^-1)of rhOSM osteogenic induction groups.Alkaline phosphatase(ALP)was stained by BCIP/NBT and the ALP activities in the cells were determined quantitatively on the 4th,7th and 14th days of osteogenic induction;Alizarin Red S staining(ARS)was performed to assess the formation of calcium nodules and the cell mineralization activities were semi-quantitatively analyzed.RT-qPCR was carried out to measure the expression levels of Runt-related transcription factor 2(Runx2)mRNA and O CN mRNA in the hUCMSCs.Results:The hUCMSCs were consistent with the identification criteria of mesenchymal stem cells(MSCs).After treatment with rhOSM for 72 h,compared with control group,the proliferation activity of hUCMSCs in 10.0μg·L^-1 rhOSM group was decreased(P<0.05);after treated for 96 h,the proliferation activities in different doses of rhOSM groups were significantly lower than that in control group(P<0.05);there were significant differences between different doses of rhOSM groups(P<0.01).On the 4th,7th and 14th days,compared with classical osteogenic induction group,the ALP activities and the expression levels of Runx2 mRNA in the hUCMSCs in different doses of rhOSM groups were increased(P<0.01).Compared with 0.1μg·L^-1 rhOSM group,the ALP activities and the expression levels of Runx2 mRNA in the hUCMSCs in 1.0 and 10.0μg·L^-1 rhOSM groups were significantly increased(P<0.05).Compared

关 键 词：抑瘤素M 脐带间充质干细胞 成骨 细胞增殖 细胞分化 

分 类 号：Q78[生物学—分子生物学]