CAJ | 학술논문

目的：对Ad－BMP－2／GFP转染兔骨髓间充质干细胞（ BMSCs ）前后的生物学特性进行观察。方法采用密度梯度离心结合贴壁培养法分离获取第10代兔BMSCs，应用流式细胞仪检测细胞表面抗原CD44、CD45、CD29的表达，用携带BMP2和GFP基因的腺病毒转染细胞。分别通过倒置荧光显微镜下观察转染前后细胞形态改变，MTT 法分析转染前后细胞增殖的情况，体外Ⅰ型胶原免疫组化染色和钙结节茜素红染色以观察转染前后细胞的成骨分化情况，Western Blot 检测转染前后细胞内目的蛋白表达。结果密度梯度离心结合贴壁培养法能获取高纯度第10代兔BMSCs ，流式细胞仪检测显示CD44、CD29阳性，CD45阴性。 Ad－BMP－2／GFP转染兔BMSCs后在荧光显微镜下观察到绿色荧光，细胞形态向成骨方向分化，在短时间内能促进兔BMSCs 的增殖（P＜0．05），Ⅰ型胶原免疫组化染色、茜素红染色均呈阳性，Western Blot显示细胞内稳定表达BMP－2目的蛋白。结论 Ad－BMP－2／GFP能成功转染兔骨髓间充质干细胞并能改变其生物学特性。
목적：대Ad－BMP－2／GFP전염토골수간충질간세포（ BMSCs ）전후적생물학특성진행관찰。방법채용밀도제도리심결합첩벽배양법분리획취제10대토BMSCs，응용류식세포의검측세포표면항원CD44、CD45、CD29적표체，용휴대BMP2화GFP기인적선병독전염세포。분별통과도치형광현미경하관찰전염전후세포형태개변，MTT 법분석전염전후세포증식적정황，체외Ⅰ형효원면역조화염색화개결절천소홍염색이관찰전염전후세포적성골분화정황，Western Blot 검측전염전후세포내목적단백표체。결과밀도제도리심결합첩벽배양법능획취고순도제10대토BMSCs ，류식세포의검측현시CD44、CD29양성，CD45음성。 Ad－BMP－2／GFP전염토BMSCs후재형광현미경하관찰도록색형광，세포형태향성골방향분화，재단시간내능촉진토BMSCs 적증식（P＜0．05），Ⅰ형효원면역조화염색、천소홍염색균정양성，Western Blot현시세포내은정표체BMP－2목적단백。결론 Ad－BMP－2／GFP능성공전염토골수간충질간세포병능개변기생물학특성。
Objective To investigate the biological characteristics of rabbit bone-marrow mesenchymal stem cells after transfection with Ad-BMP-2/GFP.Methods The 10 th generation of rabbit BMSCs were isolated and purified by density gradient centrifugation combined with adherent culture method.Cell surface antigen expression of CD44, CD45, and CD29 was detected using flow cytometry.The cells were transferred by adenovirus carrying BMP-2/GFP gene.The change of cell morphology was observed using an inverted fluorescence microscope. Cell proliferation was analyzed using MTT method. The osteoblast differentiation was studied using immunohistochemical staining for collagen type I and Alizarin red staining for calcium nodules. Targeted protein expression before and after the transfection was detected using Western blot detection.Results The 10th generation of rabbit BMSCs with high purity were successfully obtained using density gradient centrifugation and adherent culture method.Flow cytometry showed CD44 and CD29 were positive while CD45 was negative.Green fluorescence was observed in rabbit BMSCs under the fluorescence microscope after Ad-BMP-2/GFP transfection.The morphology of the cells showed osteogenic differentiation.The transfection promoted proliferation of BMSCs in a short time ( P <0.05 ) . And both type I collagen immunohistochemical staining and Alizarin red staining showed positive.Western Bloting results indicated that target protein BMP-2 expressed stably in the cells.Conclusion Rabbit BMSCs can be successfully transfected with Ad-BMP-2/GFP and the biological characteristics of the cells are significantly changed after transfection.