摘要
目的探究在体外成骨诱导环境下,辛伐他汀(simvastatin,SIM)联合岩藻多糖(fucoidan,FD)对SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向成骨细胞分化中期的影响。方法(1)从SD大鼠双侧股骨和胫骨的骨髓腔中提取原代细胞,体外培养至P3代进行细胞鉴定。鉴定方法包括细胞形态学观察、流式鉴定、成脂肪鉴定和成软骨鉴定。(2)取P3代BMSCs,分为空白组(DMEM完全培养基)和试验组(含有不同FD浓度的DMEM完全培养基)。(1)采用CCK-8法检测各组培养1、2、3天的细胞活性;(2)培养至第7天,提取细胞中的总RNA,采用qPCR法检测碱性磷酸酶(alkaline phosphatase,ALP)和骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)mRNR的表达情况。(3)培养至第7天,进行ALP定性和定量分析。综合以上结果确定FD的给药浓度。(3)取P3代BMSCs,分为对照组(OM组)、SIM组、FD组和SIM+FD组。(1)采用CCK-8法检测各组培养1、2、3天的细胞活性;(2)培养至第7天,观察细胞形态和生长状况,并拍照记录;(3)培养至第7天,提取细胞中的总RNA,采用q PCR法检测BMP-2、Ⅰ型胶原(Type I collagen,COLⅠ)、RUNX2以及ALP mRNA的表达情况;(4)培养至第7天,进行ALP定性和定量分析。结果(1)细胞鉴定。(1)细胞形态观察:细胞在培养过程中慢慢延伸并舒展开。3代以后,细胞形态基本趋于一致,成纤维样贴壁生长。(2)流式鉴定:CD29、CD90和CD44呈阳性,表达率分别为99.1%、99.5%、99.4%,CD45呈阴性,表达率为0.4%。(3)成脂肪鉴定:细胞在诱导过程中逐渐变圆,胞内出现折光性高、大小不一的脂滴,经油红O染色呈红色。(4)成软骨鉴定:细胞逐渐由长梭形变为三角形或多边形,经阿尔新兰染色,软骨基质呈蓝色,细胞核呈红色。(2)FD的给药浓度。(1)细胞活性:BMSCs的活性随培养时间和FD的浓度增加而增加。与空白组比较,差异均具有统计学意义(P<0.001)。(2)qPCR:浓度为1μg/mL的FD组BMP-2和ALP mRNA�
Objective:To investigate the effect of simvastatin(SIM)combined with fucoidan(FD)administration on the middlestage differentiation of BMSCs from SD rats to osteoblasts in the osteogenic induction environment in vitro.Methods:(1)Primary cells were extracted from the bone marrow cavity of bilateral femurs and tibias of SD rats,and cultured in vitro to P3 passage for cell identification.The identification methods included cell morphology observation,flow identification,adipogenic identification and chondrogenesis identification.(2)P3 generation BMSCs were taken and divided into blank group(DMEM complete medium)and experimental group(DMEM complete culture containing different FD concentrations base).(1)CCK-8 method was used to detect the cell activity of each group on 1,2,and 3 days of culture.(2)To the 7th day of culture,the total RNA in the cells was extracted,and the qPCR method was used to detect the expressions of mRNR in Alkaline phosphatase(ALP)and bone morphogenetic protein-2(BMP-2)..(3)To the 7th day,the qualitative and quantitative analysis of ALP were carried out.Based on the above results,the administration concentration of FD was determined.(3)P3 generation BMSCs were taken and divided into control group(OM group),SIM group,FD group and SIM+FD group.(1)CCK-8 method was used to detect the cell viability of each group for 1,2,and 3 days of culture.(2)The cell morphology and growth status were observed on the 7th day of culture,and photographed and recorded.(3)To the 7th day of culture,the total cells in the cells were extracted.RNA,qPCR method was used to detect the expressions of BMP-2,Type I collagen(COLⅠ),RUNX2 and ALP mRNA.(4)To the 7th day of culture,the qualitative and quantitative analyses of ALP wwere carried out.Results:(1)(1)Observation of cell morphology showed that cells slowly extended and stretched out during the culture process.After 3 generations,the shape of the cells was basically the same,and the fibroblastlike growth adhered to the wall.(2)The detection of flow cytometry showed that C
作者
王莹
詹玉林
WANG Ying;ZHAN Yulin(School of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China;Department of Orthopedics,Sixth People’s Hospital Affiliated to Shanghai Jiaotong University,Shanghai 201306 China)
出处
《山东第一医科大学（山东省医学科学院）学报》
CAS
2022年第12期897-905,共9页
Journal of Shandong First Medical University ＆ Shandong Academy of Medical Sciences
基金
上海市卫生计生委重点项目(201640004)。
关键词
辛伐他汀
岩藻多糖
骨髓间充质干细胞
成骨诱导
simvastatin
fucoidan
bone marrow mesenchymal stem cells
osteogenic induction
