牙周膜干细胞向成骨细胞方向分化实验  

Experiment on inducing periodontal ligament stem cell to differentiate into osteoblasts in vitro

作　　者：贺慧霞[1] 刘洪臣[2] 郭宏[1] 杨洋[3] 刘一涵[2] 王东胜[2] 鄂玲玲[2] 

机构地区：[1]解放军总医院老年口腔病科,副主任医师北京100853 [2]解放军总医院口腔医学研究所,主任主任医师教授北京100853 [3]解放军总医院口腔内科,主治医师北京100853

出　　处：《中华老年口腔医学杂志》2011年第3期129-133,共5页Chinese Journal of Geriatric Dentistry

基　　金：国家自然科学基金资助项目(编号:81070833);中国博士后科学基金(编号:20070420077);中国博士后科学基金特别资助(编号:200801061)

摘　　要：目的:探讨人牙周膜干细胞(Periodontalligament stem cell,PDLSCs)向成骨细胞定向分化潜能,从细胞、分子和基因水平分析矿化诱导过程中细胞形态、功能变化。方法:将免疫磁珠分离的人PDLSCs矿化诱导,通过倒置显微镜观察诱导细胞形态变化,ELISA法检测诱导细胞碱性磷酸酶活性(ALPase)、RT-PCR、Western blot及免疫化学染色分析其成骨相关基因、蛋白表达变化,茜素红染色法检测其矿化结节形成情况来诱导细胞做对照。结果:矿化诱导14d,细胞形态呈成骨样短突起、多角形改变,诱导7d、14d ALPase活性显著增强。21d,诱导细胞高丰度表达骨桥素(OPN)、骨钙素(OCN)、牙骨质附着蛋白(CAP)及I型胶原(COLⅠ)mRNA,而对照组只有COLⅠmRNA弱表达。同时,Western blot及免疫化学染色均显示诱导细胞分泌骨涎蛋白(BSP)。21d,仅诱导组形成大量矿化结节。结论:人PDLSCs在矿化液诱导下可向成骨细胞分化,具有成骨细胞的形态、表型和功能特点,有望成为牙周及种植体周围骨缺损修复再生的理想的种子细胞。Objective:To explore the capability of human periodontal ligament stem cells(PDLSCs) differentiating into osteoblasts in vitro and to determine their changes in cell morphology and function during differentiation at cellular level,molecular level and genetic level,respectively.Methods: PDLSCs isolated by magnetic-activated cell selection were treated continuously with mineralization solution for 21d.Then cell morphology,osteoblast related proteins and genes markers were analyzed by inverted contrast microscope,immunocytochemical staining,Western blot and RT-PCR,respectively.The ALPase activity was detected at day 7 and day 14 and alizarin red staining was also performed at day 21,the non-induced cell was used as a control.Results:The induced cells differentiated into osteoblast-like cells with polygon in shape after induction for 21 days.Compared with the control group,the ALPase activity of the cells in induced group were significantly higher at day 7 and day 14 and was positively expressed osteogenesis related genes of cement attachment protein(CAP),osteocalcin(OCN),osteopontin(OPN) and Collengen Ⅰ(Col I)mRNA.Moreover,the induced cell positively expressed bone sialoprotein(BSP) and Col I,protein markers of bone cell.In addition,only the induced cell could form mineralized nodes as showed by alizarin red staining.Conclusion:Human PDLSCs have the potential of differentiation into osteoblasts under specific culture condition,and the differentiated cells exhibited characteristics of osteoblast cells both in cell morphology and in cell functions.This study suggests that PDLSCs can be used as a potent seed cell for stem cell-based periodontal and peri-implant bone tissue engineering.

关 键 词：牙周膜干细胞 成骨 矿化液 诱导 

分 类 号：R78[医药卫生—口腔医学]