CAJ | 학술논문

目的观察绿色荧光蛋白( GFP)标记的兔骨髓间充质干细胞( BMSCs)成骨诱导后形态学改变和体外骨生成中矿化钙结节的形成,并结合扫描电镜和X射线能谱分析技术( SEM/EDS)对矿化钙结节元素进行定量能谱分析。方法<br>　　用携带GFP基因的腺病毒转染兔BMSCs进行示踪标记,并诱导细胞向成骨方向分化,通过倒置荧光显微镜、钙结节茜素红染色观察成骨诱导后细胞形态改变和矿化钙结节的形成,结合SEM/EDS技术观测矿化钙结节的表面微观结构及其元素构成。结果腺病毒介导GFP基因( Ad-GFP )转染兔 BMSCs后在荧光显微镜下观察到绿色荧光,经成骨诱导后细胞形态向成骨方向分化,并形成不透光的矿化钙结节,钙结节茜素红染色见红色矿化结节,SEM下见矿化钙结节散布于细胞中,细胞重叠生长,分泌基质旺盛。 EDS分析显示矿化钙结节主要组成元素与正常骨组织相同,其钙磷比(Ca/P)为1.55,接近正常骨组织钙磷比1.49。结论 Ad-GFP能成功转染并标记兔BMSCs,成骨诱导后BMSCs向成骨方向分化,具备较好的骨生成能力,BMSCs体外矿质化成骨过程与体内基本相同。
목적관찰록색형광단백( GFP)표기적토골수간충질간세포( BMSCs)성골유도후형태학개변화체외골생성중광화개결절적형성,병결합소묘전경화X사선능보분석기술( SEM/EDS)대광화개결절원소진행정량능보분석。방법<br>　　용휴대GFP기인적선병독전염토BMSCs진행시종표기,병유도세포향성골방향분화,통과도치형광현미경、개결절천소홍염색관찰성골유도후세포형태개변화광화개결절적형성,결합SEM/EDS기술관측광화개결절적표면미관결구급기원소구성。결과선병독개도GFP기인( Ad-GFP )전염토 BMSCs후재형광현미경하관찰도록색형광,경성골유도후세포형태향성골방향분화,병형성불투광적광화개결절,개결절천소홍염색견홍색광화결절,SEM하견광화개결절산포우세포중,세포중첩생장,분비기질왕성。 EDS분석현시광화개결절주요조성원소여정상골조직상동,기개린비(Ca/P)위1.55,접근정상골조직개린비1.49。결론 Ad-GFP능성공전염병표기토BMSCs,성골유도후BMSCs향성골방향분화,구비교호적골생성능력,BMSCs체외광질화성골과정여체내기본상동。
Objective To observe the morphological changes of rabbit bone-marrow mesenchymal stem cells ( BM-SCs ) after osteogenic induction and calcified plaque formation of osteogenesis of rabbit BMSCs labeled by green flu-orescent protein( GFP) in vitro and quantitatively analyze the elements of calcified plaque by scanning electron mi-croscope and energy dispersive spectrometer( SEM/EDS) . Methods The rabbit BMSCs were traced by adenovirus with GFP( Ad-GFP) gene, and then cells were induced differentiation to osteogenesis. Inverted fluorescence micro-scope and alizarin red staining were applied to observe morphological changes of cells after transfection and calcified plaque formation and SEM/EDS were employed to study the surface microstructure and element composition of cal-cified plaque. Results Fluorescence microscope showed rabbit BMSCs after Ad-GFP transfection gave green light and the cell morphology differentiated to osteogenic accompanied with opaque mineralized calcium nodule. Alizarin red staining exhibited red mineralized nodules. From SEM, punctate mineralization calcium nodules scattered a-mong cells and cells grew overlapply strongly secreted matrix. EDS confirmed that the main elements of mineralized calcium nodules were analogous to normal bone tissue and ratio of calcium to phosphorus (Ca/P) was 1. 55, close to normal ratio 1. 49 . Conclusion Rabbit BMSCs can be successfully transfected and labeled by Ad-GFP and trend to osteogenic differentiation after osteogenesis induction. BMSCs have a preferable osteogenic capability and the process of biological mineralization in vitro is the same with in vivo.