雌激素缺乏对大鼠颌骨骨髓间充质干细胞增殖及成骨向分化能力的影响  

Effect of estrogen deficiency on the proliferation and osteogenic differentiation potential of mandibular bone marrow stromal cells

作　　者：王燕萍[1] 宋卫健[2] 景双林[3] 于金华[3] 

机构地区：[1]苏州大学附属口腔医院·苏州口腔医院牙体牙髓病科,江苏215005 [2]苏州大学附属口腔医院·苏州口腔医院牙体牙髓病科 [3]南京医科大学口腔医学研究所南京医科大学附属口腔医院牙体牙髓病科 ,210029

出　　处：《中华口腔医学杂志》2014年第10期619-624,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81371144）;江苏省自然科学基金（BK20131392）

摘　　要：目的 探讨雌激素缺乏对大鼠颌骨骨髓间充质干细胞（mandibular bone marrow stem cells,mBMSC）增殖及成骨向分化能力的影响,以期为组织工程颌骨再生及种子细胞选择提供实验室依据.方法 将10只8周龄成年雌性SD大鼠用抽签法随机分为假手术组及卵巢去势组（去势组）,每组5只.全麻下将去势组大鼠双侧卵巢摘除,假手术组行相同切口,保留双侧卵巢.1个月后处死大鼠,分离下颌骨,全骨髓培养法分离培养两组mBMSC.采用甲基噻唑基四唑（methyl thiazolyl tetrazolium,MTT）法、碱性磷酸酶（alkaline phosphotase,ALP）活性检测、茜素红染色、实时反转录PCR和蛋白质印迹法检测雌激素缺乏对mBMSC增殖及成骨向分化能力的影响.结果 MTT生长曲线显示去势组mBMSC的增殖能力显著低于假手术组.两组细胞培养3d后去势组ALP活性[（0.710±0.011） Sigma单位/蛋白]显著低于假手术组[（1.512±0.021） Sigma单位/蛋白],差异有统计学意义（P＜0.01）.14d时茜素红染色结果显示去势组mBMSC形成的矿化结节较假手术组少,钙离子浓度定量检测结果显示去势组mBMSC钙结节中的钙离子含量[（0.433±0.045） μg/g]显著低于假手术组[（1.453±0.131） μg/g]（P＜0.01）.实时反转录PCR及蛋白质印迹法检测结果显示,去势组mBMSC在培养7 d后,各项骨向分化相关指标的表达均显著低于假手术组（P＜0.05）.结论 雌激素缺乏显著降低大鼠mBMSC体外增殖及成骨向分化能力.Objective To determine the effects of estrogen deficiency on the proliferation and osteogenic differentiation of mandibular bone marrow stromal cells（mBMSC）.Methods Ten 8-week-old female SD rats were randomly divided into two groups,ovariectomized group（OVX,n=5） and sham-operation group（n=5）.All rats were anesthetized and both ovaries of OVX-rats were gently removed.Sham-operation rats were treated with the same incisions to expose the ovaries but without any hurt to them.One month after the operation,the mandibular bones were gently separated and mBMSC were isolated.Methyl thiazolyl tetrazolium（MTT） assay,alkaline phosphotase（ALP） activity,alizarin red staining,real-time reverse transcription（RT）-PCR and Western blotting were respectively used to examine the proliferative activity and osteogenic potential of mBMSC.Results MTT results showed that OVX-mBMSC exhibited the decreased proliferative activity as compared with Sham-mBMSC.ALP activity of OVX-mBMSC[（0.710±0.011） Sigma unit/protein] was lower than that of Sham-mBMSC[（1.512±0.021） Sigma unit/protein]（P〈0.01）.Alizarin red staining showed that OVX-mBMSC formed less calcified nodules than Sham-mBMSC.Ca2＋ concentration analysis showed Ca2＋ of OVX-mBMSC[（0.433±0.045） μg/g] was less than Sham-mBMSC[（1.453±0.131） μg/g]（P〈0.01）.Real-time RT-PCR and Western blotting results showed that the expression of osteogenic markers （Alp,Runx2/RUNX2,Osx/OSX,Ocn/OCN） in OVX-mBMSC was significantly inhibited as compared with Sham-mBMSC（P〈0.05）.Conclusions Estrogen deficiency significantly inhibits the proliferation and osteogenic capacity of mBMSC.

关 键 词：雌激素类 间充质干细胞 细胞增殖 细胞分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]