摘要
目的评价牙本质涎磷蛋白(DSPP)转基因修饰骨髓间质干细胞(BM-MSC)后,对BM- MSC生物学特性及DSPP表达的影响。方法构建含小鼠DSPP基因的真核表达载体pcDNA3．1 (+)／DSPP,用脂质体介导转染大鼠BM-MSC;RT-PCR检测转染后细胞的Pax-9和DMP-1基因表达情况;检测转染细胞矿化诱导后Von Kossa钙盐染色计算单位面积钙结节形成率。结果成功构建DSPP真核表达载体pcDNA3．1(+)／DSPP,酶切后得到3．0 kb和5．4 kb的片段,与回收的目的基因和载体基因片段大小相符;经转染BM-MSC后24 h可见DSPP基因表达,48 h后可见有Pax-9基因表达,无DMP-1基因表达;转基因后的BM-MSC免疫组化染色显示DSPP阳性;转染细胞矿化诱导后钙结节形成率高于未转染细胞。结论BM-MSC转基因表达DSPP能够增强其矿化能力,并诱导牙齿发育相关基因的表达,提示DSPP可能在牙齿发育早期具有一定作用。
Objective To evaluate the expression of dentin sialophosphoprotein （DSPP） in transfected rat bone marrow mesenchymal stem cells （BM-MSC） and the influence of the transfection. Methods Plasmid containing mice dentin DSPP was constructed by using the cytomegalovirus （CMV） promoter and then transfected the cultured BM-MSC by lipofectamine; The expression of Pax-9 and dentin matrix protein 1 （ DMP1 ） gene of transfected BM-MSC were detected by RT-PCR. The expression of DSPP was examined by immunocytochemical staining, and the formation ratio of mineralized nodules of transfected BM-MSC was compared with untransfected ones after mineralized induction. Results The constructed pcDNA3.1 （＋）/mDSPP could produced 3. 0 kb and 5.4 kb fragments , DSPP gene and Pax9 gene were expressed 24 h and 48 h respectively , after BM-MSC were transfected Pax-9 gene was exprssed , but DMP1 gene was not; Immunohistochemical staining shewed that DSPP was positive in transfected BM-MSC; The formation ratio of mineralized nodules of transfected BM-MSC was higher than that of untransfected ones after mineralized induction. Conclusions The expression of mice DSPP in BM-MSC by gene transfection can induce the expression of tooth development-associated gene Pax9 and enhance the formation of mineralized nodules, which suggests that DSPP gene might induce odontogenic differentiation of BM-MSC.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2006年第7期426-429,共4页
Chinese Journal of Stomatology
基金
国家自然科学基金资助项目(30572051)
国家重大基础研究前期研究专项基金资助项目(2002CCC00700)
关键词
磷蛋白类
干细胞
间质细胞
Phosphoproteins
Stem cells
Stromal cells
