摘要
目的探讨胰岛素样生长因子-1(IGF-1)基因修饰对诱导脂肪干细胞(ADSCs)向软骨细胞分化的实验研究。方法收集大鼠收集其大网膜、肾周脂肪囊以及睾丸周边的脂肪组织,分离大鼠ADSCs后,采用免疫荧光检测所得细胞的表面抗原来鉴定细胞,同时将ADSCs体外培养,细胞实验分为正常组、对照组、实验组。将pcDNA3.1-IGF-1和pcDNA3.1-IGF-1-NC分别通过阳离子脂质介导法转染至实验组、对照组细胞,显微镜下观察各组细胞形态变化,依次使用茜素红、油红O、甲苯胺蓝染色、免疫荧光、蛋白质免疫印迹试验(Western blot)检测各组细胞向成骨、成脂、成软骨分化能力、Ⅰ型胶原蛋白(ColⅠ)、Ⅱ型胶原蛋白(ColⅡ)的表达、ColⅠ、ColⅡ和IGF-1蛋白的表达水平。结果免疫荧光结果显示,提取细胞的抗原决定簇CD29、CD44呈阳性反应,CD14、CD34呈阴性,所提取细胞为大鼠ADSCs。正常组和对照组贴壁生长良好,形态清晰,细胞多为长梭形或角形。实验组细胞呈现旋涡状生长,细胞增殖较正常组快,细胞中央包体突起较多,有形成结节的趋势。与正常组相比,过表达IGF-1基因后,实验组细胞的成骨、成脂、成软骨分化能力明显增强,ColⅠ、ColⅡ荧光强度明显增强,ColⅠ、ColⅡ和IGF-1蛋白表达水平明显升高,差异均具有统计意义(P<0.05),正常组细胞和对照组细胞相比,差异无统计意义(P>0.05)。结论IGF-1的基因修饰能明显促进大鼠脂肪充质干细胞向软骨细胞的分化。
Objective To investigate effect of insulin-like growth factor-1(IGF-1)gene modification to induce adipase derived stem cells(ADSCs)to differentiate into chondrocytes.Methods Rats were collected and collected from the omentum,the perirenal fat sac,and the adipose tissue around the testis.After the rat ADSCs were isolated,the surface antigens of the cells obtained by immunofluorescence detection to identify the cells.culture cells in vitro.The cell experiments were divided into normal group,control group and experimental group.The pcDNA3.1-IGF-1 and pcDNA3.1-IGF-1-NC were transfected into cells of the experimental group and control group,respectively.The microscope was use to Observe the morphological changes of cells in each group.Alizarin red,oil red O,toluidine blue staining,immunofluorescence,and Western blot were used to detect osteogenic,adipogenic,chondrogenic differentiation ability of cells in each group,the expression of ColⅠ,ColⅡ,the expression levels of ColⅠ,ColⅡand IGF-1 protein.Results The immunofluorescence results showed that the antigenic determinants CD29 and CD44 of the extracted cells were positive and CD14 and CD34 were negative.The extracted cells were rat ADSCs.The normal group and the control group had good adherent growth,clear morphology,and most of the cells were long spindle or angular.The cells in the experimental group showed vortex-like growth,the cells proliferated faster than the normal group,and the central inclusions of the cells had more protrusions,and there was a tendency to form nodules.Compared with the normal group,Compared with the normal group,the osteogenic,adipogenic,and chondrogenic differentiation ability of cells in the experimental group was significantly enhanced.The fluorescence intensity of ColⅠand ColⅡis obviously enhanced.and the expression levels of ColⅠ,ColⅡ,and IGF-1 protein were significantly increased.The differences were statistically significant(P<0.05).There was no statistical difference between the normal group and the control group(P>0.
作者
史正亮
张华
范志勇
李秋童
李身泰
袁鹤
王斌
SHI Zheng⁃liang;ZHANG Hua;FAN Zhi⁃yong;LI Qiu⁃tong;LI Shen⁃tai;YUAN He;WANG Bin(Department of Orthopedics,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)
出处
《解剖学研究》
CAS
2020年第5期435-441,共7页
Anatomy Research
关键词
胰岛素样生长因子-1
脂肪干细胞
软骨细胞分化
大鼠
Insulin-like growth factor-1
Adipase derivedstem cells
Chondrocyte differentiation
Rat
