神经生长因子对2型糖尿病小鼠骨髓基质细胞体外成骨能力的影响  

Effect of nerve growth factor on osteogenic potential of type 2 diabetic mice bone marrow stromal cell in vitro

作　　者：崔国胜[1] 曾剑玉[1] 张婧 卢燃[1] 

机构地区：[1]首都医科大学口腔医学院修复科,100050

出　　处：《中华口腔医学杂志》2018年第2期97-102,共6页Chinese Journal of Stomatology

基　　金：北京市卫生系统高层次卫生技术人才培养计划（2011-3-075）

摘　　要：目的探讨神经生长因子（nerve growth factor，NGF）对2型糖尿病小鼠骨髓基质细胞（bone marrow stromal cell，BMSC）增殖、成骨向分化及矿化能力的影响，为NGF的临床应用提供依据。方法选择8周龄雄性2型糖尿病db/db小鼠3只和正常C57BL/6J小鼠2只，全骨髓贴壁法分离培养股骨BMSC。用糖尿病小鼠BMSC（糖尿病组）和正常小鼠BMSC（正常组）进行BMSC成骨能力比较实验（实验一）；用糖尿病小鼠BMSC进行NGF对糖尿病小鼠BMSC成骨能力影响实验（实验二），分为糖尿病对照组、NGF组和K252a＋NGF组[K252a为NGF高亲和力受体酪氨酸激酶A（tyrosine kinase，TrkA）的特异性阻断剂]；K252a＋NGF组加K252a 30 min后加NGF；NGF组仅加NGF，糖尿病对照组两者均不添加。甲基噻唑基四唑法检测实验一基础培养1、3、5、7 d和实验二基础培养1、2、3 d的细胞增殖情况；成骨诱导3、5、7 d后检测碱性磷酸酶（alkaline phosphatase，ALP）水平；成骨诱导14 d后茜素红染色观察矿化结节形成情况。每项实验每组均设置5个复孔，所有实验重复3次。结果基础培养3、5、7 d，糖尿病组BMSC增殖能力均显著高于相同时间点正常组（P〈0.05）；成骨诱导3、5、7 d，糖尿病组ALP水平均显著低于相同时间点正常组（P〈0.05）；成骨诱导14 d，糖尿病组矿化结节数量[（23.1±6.4）个/视野]显著少于正常组[（36.9±7.9）个/视野]（P〈0.05）。基础培养1、2、3 d，糖尿病对照组、NGF组和K252a＋NGF组BMSC增殖差异无统计学意义（P〉0.05）；成骨诱导3、5 d，NGF组ALP水平均显著高于糖尿病对照组（P〈0.05）；成骨诱导3、5、7 d，K252a＋NGF组ALP水平均显著低于NGF组（P〈0.05）和糖尿病对照组（P〈0.05）；成骨诱导14 d，NGF组矿化结节数量[（45.2±6.8）个/视野]显著多于糖尿病对照组[（23.1±6.4）个/视野]（P〈0.05），K252a＋NGF组矿化结节数量[（18.0±4.5）�Objective To study the effects of nerve growth factor （NGF） on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell （BMSC）, providing basis for clinical application of NGF.Methods Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups： diabetic control group, NGF group, and K252a＋NGF group [K252a was the inhibitor of tyrosine kinase A （TrkA）, which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a＋NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a＋NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase （ALP） at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times.Results The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d （P〈0.05）. After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group （P〈0.05）. After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [（23.1±6.4） nodule/field] were significantly lower than that of normal group [（36.9±7.9） nodule/field]（P〈0.05）. At 1, 2 and 3 d, BMSC proliferati

关 键 词：糖尿病 2型 神经生长因子 骨髓祖代细胞 酪氨酸激酶A 

分 类 号：R587.1[医药卫生—内分泌]