CAJ | 학술논문

目的探讨转录因子特殊富含AT序列结合蛋白2(special AT-rich binding protein 2,SATB2)在骨髓基质细胞(bone marrow stromal cells,BMSC)成骨分化中的作用,以期为牙组织工程筛选种子细胞及调控因子提供实验依据.方法采用密度梯度离心与贴壁培养相结合的方法,分离并鉴定BMSC;构建SATB2表达载体,转染BMSC,设立转染组(SATB2组)、转染空质粒组(Mock组)及未转染组(对照组),蛋白质印迹法及反转录聚合酶链反应(RT-PCR)检测SATB2蛋白及mRNA的表达,分析SATB2在成骨分化过程中对成骨因子(Runx2、Osx、Atf4及骨涎蛋白)表达的影响.结果 BMSC在体外分离培养后形态呈长梭形或多角形;流式细胞仪分析结果显示第3代BMSC细胞CD29呈阳性表达,CD34呈阴性表达;生长曲线呈S形.satb2基因成功转染至BMSC后,矿化结节明显增多,SATB2组IA值为125974±241,对照组IA值为178 486±406;且迁移速率提高,8 h时SATB2组划痕宽度为(0.72±0.01) mm,对照组为(0.83±0.03) mm.SATB2组Runx2、Osx、Atf 4及骨涎蛋白的表达与对照组相比均增高.结论获得了均一性较好的牙组织工程种子细胞;明确了SATB2可有效诱导BMSC的成骨分化,为建立具有潜在临床应用价值的种植牙骨整合研究模型提供了实验依据.
목적 탐토전록인자특수부함AT서렬결합단백2(special AT-rich binding protein 2,SATB2)재골수기질세포(bone marrow stromal cells,BMSC)성골분화중적작용,이기위아조직공정사선충자세포급조공인자제공실험의거.방법 채용밀도제도리심여첩벽배양상결합적방법,분리병감정BMSC;구건SATB2표체재체,전염BMSC,설립전염조(SATB2조)、전염공질립조(Mock조)급미전염조(대조조),단백질인적법급반전록취합매련반응(RT-PCR)검측SATB2단백급mRNA적표체,분석SATB2재성골분화과정중대성골인자(Runx2、Osx、Atf4급골연단백)표체적영향.결과 BMSC재체외분리배양후형태정장사형혹다각형;류식세포의분석결과현시제3대BMSC세포CD29정양성표체,CD34정음성표체;생장곡선정S형.satb2기인성공전염지BMSC후,광화결절명현증다,SATB2조IA치위125974±241,대조조IA치위178 486±406;차천이속솔제고,8 h시SATB2조화흔관도위(0.72±0.01) mm,대조조위(0.83±0.03) mm.SATB2조Runx2、Osx、Atf 4급골연단백적표체여대조조상비균증고.결론 획득료균일성교호적아조직공정충자세포;명학료SATB2가유효유도BMSC적성골분화,위건립구유잠재림상응용개치적충식아골정합연구모형제공료실험의거.
Objective To investigate the role of transcription factor special AT-rich binding protein 2(SATB2) in the osteoblasts differentiation of bone marrow stromal cells (BMSC) in vitro. Methods Rats bone marrow stromal cells were isolated by Percoll sedimentation and the cells were placed and allowed to attach for three times. After passages,expression plasmid pBABE-hygro-satb2 was constructed, then transfected into BMSC. BMSCs were inoculated in conditioned medium and osteogenic factors were detected by western blotting and reverse transcription polymerase chain reaction(RT-PCR). Results The morphological observation of BMSC showed either spindle or polygonal pattern. The cellular phenotypic marker of the third passage was CD29 positive and CD34 negative. The growth curve possessed "S" pattern. The intensity of calfilication in BMSC was higher in SATB2 transfection group (IA value 125974±241) than that in the control groups (IA value 178486±406). Moreover, cell migration rate increased in SATB2 transfection group [width of scratch (0.72±0.01) mm] compared with control group[width of scratch (0.83±0.03) mm]. In addition, the mRNA expression of osteogenic factors runt-related transcription factor 2,Osterix, activating transcription factor 4,integrin-binding sialoprotein were upregulated. Conclusions Cells cultured with this method have general biological characteristics and osteogenic differentiation potential in vitro. SATB2 can promote osteoblasts differentiation of BMSC.