CAJ | 학술논문

目的：体外培养兔骨髓间充质干细胞（BMSCs）并观察其生物学特性，通过腺病毒介导人骨形态发生蛋白-2（hBMP-2）和EGFP基因转染兔BMSCs后，观察BMP-2和EGFP的表达情况。方法通过密度梯度离心联合贴壁法，分离、培养兔BM-SCs ，流式细胞仪检测细胞周期和表面标记，体外诱导兔BMSCs向成骨细胞方向分化。转染后，免疫细胞化学染色及蛋白质印迹法（Western blot）检测外源基因的表达。结果兔BMSCs周期约有56．84％处于G1期；CD44表达阳性，CD45表达阴性；经成骨细胞诱导后，Ⅰ型胶原免疫组织化学染色阳性，茜素红染色阳性。转染后，Western blot及免疫细胞化学染色显示细胞内BM P-2有较强的表达。结论成功分离、培养兔BMSCs ，并鉴定其具有成骨分化能力；构建的 Ad-BMP-2/EGFP 可高效转染兔BMSCs ，并能稳定表达BM P-2目的基因。
목적：체외배양토골수간충질간세포（BMSCs）병관찰기생물학특성，통과선병독개도인골형태발생단백-2（hBMP-2）화EGFP기인전염토BMSCs후，관찰BMP-2화EGFP적표체정황。방법통과밀도제도리심연합첩벽법，분리、배양토BM-SCs ，류식세포의검측세포주기화표면표기，체외유도토BMSCs향성골세포방향분화。전염후，면역세포화학염색급단백질인적법（Western blot）검측외원기인적표체。결과토BMSCs주기약유56．84％처우G1기；CD44표체양성，CD45표체음성；경성골세포유도후，Ⅰ형효원면역조직화학염색양성，천소홍염색양성。전염후，Western blot급면역세포화학염색현시세포내BM P-2유교강적표체。결론성공분리、배양토BMSCs ，병감정기구유성골분화능력；구건적 Ad-BMP-2/EGFP 가고효전염토BMSCs ，병능은정표체BM P-2목적기인。
Objective To culture the rabbit bone marrow derived mesenchymal stem cells (BMSCs) ,and to observe their biologi-cal characteristics in vitro and the expression of BMP2 and EGFP after Ad-EGFP-BMP2 infected on them .Methods To isolate and cultivate BMSCs by density gradient centrifugation and adherent culture in vitro ;the cell cycle and the surface marks were detected by flow cytometer ;rabbit BMSCs were differentiated into the direction of osteogenetic cells by in vitro induction .After transfection , the expression of exogenous gene in the cells was detected by the immunocytochemical staining and Western blot .Results About 56 .84% of rabbit BMSCs cell cycle was in the G1 phase;the D44 expression was positive and the CD45 expression was negative ;af-ter induction by osteogenetic cells ,the Ⅰtype collagen immunohistochemical staining was positive and the alizarin red staining was positive .After transfection ,the strong expression of BMP2 and EGFP in cells was showed by Western blot and the immunocyto-chemical staining .Conclusion rabbit BMSCs are successfully isolated ,cultured and identified to have the ability of osteogenetic dif-ferentiation ;the structured Ad-BMP-2/EGFP can efficiently transfected rabit BMSCs and stably express the target gene of BMP 2 .