胰岛素生长因子对骨髓基质细胞增殖和向成骨细胞分化的调节  

Regulatory effects of insulin growth factor-1 on the proliferation and differentiation of marrow stromal cells into osteoblasts

作　　者：魏波[1] 陈日景[1] 

机构地区：[1]广东医学院附属医院,广东省湛江市524001

出　　处：《中国组织工程研究与临床康复》2007年第24期4773-4776,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东医学院博士启动基金项目(200205/B0503)~~

摘　　要：目的:观察胰岛素生长因子对骨髓基质细胞向成骨细胞分化的调节作用。方法:实验于2005-11/2006-04在广东医学院附院中心实验室进行。①实验材料:重组胰岛素生长因子(GIBCO),DEME培养基(GIBCO),胰蛋白酶(GIBCO),胎牛血清(杭州四季青),二甲基亚砜(sigma),四甲基偶氮噻唑盐(sigma),碱性磷酸酶试剂盒(长城临床试剂公司),骨钙素试剂盒(天津九鼎)。SD大鼠由广东医学院动物部提供。②实验方法:骨髓基质细胞的分离:将SD大鼠处死后,无菌条件下取出动物的双侧股骨和胫骨,分离培养骨髓基质细胞。骨髓基质细胞成骨诱导条件:取第2代骨髓基质细胞,用含体积分数为0.1胎牛血清、50mg/L维生素C、10mmol/Lβ-甘油磷酸钠、10-8mol/L地塞米松、100U/mL青霉素、100U/mL链霉素成骨诱导培养液培养,置37℃、体积分数为0.05CO2饱和湿度培养箱内孵育。倒置显微镜观察细胞生长状况。③实验分组:将第3代骨髓基质细胞随机分为对照组和实验组。细胞增殖实验:取第3代生长良好的骨髓基质细胞,密度调整至4×106L-1。实验组加入含1,10,20μg/L胰岛素生长因子诱导培养液,对照组加不含胰岛素生长因子诱导培养液。培养1,3,5,7d后采用MTT法检测细胞增殖水平。用碱性磷酸酶试剂盒在全自动生化分析仪上测定细胞内碱性磷酸酶活性。④实验评估:采用RI测定上清液中骨钙素含量。观察胰岛素生长因子对骨髓基质细胞增殖和向成骨细胞方向分化的调节作用。结果:①MTT法测骨髓基质细胞增殖情况:与对照组相比,实验组具有明显促进骨髓基质细胞增殖作用(吸光度值:1d,0.442±0.002,0.498±0.009,0.546±0.004,0.553±0.005;3d,0.571±0.008,0.604±0.007,0.682±0.006,0.694±0.009;5d,0.623±0.003,0.659±0.004,0.793±0.008,0.807±0.007;7d,0.792±0.008,0.810±0.006,0.912±0.003,0.923±0.004,P<0.05)。20μg/L胰岛素生长因子与10μg/L胰岛素生长因子组的作用效果差异无显著AIM： To investigate the effects of insulin growth factor （IGF-1） on the differentiation of marrow stromal cells （MSCs） into osteoblasts. METHODS： The experiment was carded out in the central laboratory of Guangdong Medical College from November 2005 to April 2006. ①The materials in the experiment included recombinant insulin growth factor, DEME culture medium, and trypsin （GIBCO）, fetal bovine serum （Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.）, dimethyl sulffoxide （DMSO, Sigma）, methyl thiazolyl tetrazolium （MTT, Sigma）, alkaline phosphatase kit （ALP, Great Wall Clinical Reagent Company）, and osteocalcin kit （Tianjin Jiuding）. ②The SD rats （animal center of Guangdong Medical College） were sacrificed, and their bilateral femur and tibia were separated sterUely to isolate and culture the MSCs. The second generation MSCs were cultured in osteogenetic cultured solution containing 0.1 volume fraction fetal bovine serum, 50 mg/L vitamin C, 10 mmol/L beta-sodium glycerophosphate, 10^-8 mol/L dexamethasone, 100 U/mL penicillin and 100 U/mL streptomycin and put in the saturated humidity incubator with 0.05 volume fraction CO2 at 37 %. The cell growth condition was observed under inverted microscope.③Then the third-generation MSCs were randomly divided into control group and experimental group. After the cell concentration was adjusted to 4×10^8 L^-1, the experimental group was added 1, 10, and 20μg/L IGF-1, but the control group was not. One, three, five, and seven days later, the proliferation of MSCs was detected by MTT method, and the activity of ALP was measured according to the kit by the automatic biochemistry analyzer.④The level of osteocalcin in the supernatant fluid was measured by RI, and the regulatory effects of IGF-1 on the proliferation and differentiation of MSCs into osteoblasts were observed. RESULTS： ①MTT results suggested that compared with the control group, MSCs exposed to IGF-1 proliferated significantly （absorbance value： da

关 键 词：胰岛素生长因子 骨髓基质细胞 成骨细胞 分化 

分 类 号：R392.1[医药卫生—免疫学]