环氧化酶2基因沉默不影响人骨髓间充质干细胞定向分化的能力.doc

环氧化酶2基因沉默不影响人骨髓间充质干细胞定向分化的能力赫 天1，张海宁1，项金瑜2，刘相萍3，邓继红4，王英振1青岛大学医学院附属医院，1山东省创伤骨科研究所，关节外科，3中心实验室，山东省青岛市 266003，2青岛大学附属烟台毓璜顶医院肿瘤科，山东省烟台市 264000；4双鸭山市人民医院检验科，黑龙江省双鸭山市 55100Cyclooxygenase 2 silencing has no effects on oriented differentiation of human bone marrow mesenchymal stem cellsHe Tian1, Zhang Hai-ning1, Xiang Jin-yu2, Liu Xiang-ping3, Deng Ji-hong4, WangYing-zhen11Department of Joint Surgery, Shandong Institute of Orthopaedics and Traumatology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China; 2Department of Oncology, Yantai Yuhuangding Hospital, Yantai 264000, Shandong Province, China; 3Central Laboratory, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China; 4Department of Clinical Laboratory, Shuang Yashan Peoples Hospital, Shuangyashan 155100, Heilongjiang Province, China摘要背景：目前环氧化酶2基因沉默在干细胞中的研究还处在初级阶段。目的：构建携带环氧化酶2基因沉默的慢病毒载体，观察其在人骨髓间充质干细胞中的表达，以及其定向分化潜能变化情况。方法：应用重组慢病毒技术构建携带沉默环氧化酶2基因和绿色荧光蛋白的慢病毒载体，并用其转染体外培养第3代的人骨髓间充质干细胞，以无目的基因的慢病毒转染人骨髓间充质干细胞的实验组和未做处理的人骨髓间充质干细胞分别作为阴性对照组和空白组进行对比。转染后7 d分别提取各组细胞的总RNA和蛋白检测环氧化酶2表达量。对环氧化酶2基因沉默的人骨髓间充质干细胞和正常人骨髓间充质干细胞定向诱导，观察环氧化酶2基因沉默对人骨髓间充质干细胞成脂、成骨、成软骨能力的影响。结果与结论：慢病毒转染3 d后，荧光显微镜下可见人骨髓间充质干细胞发出绿色荧光，阴性对照组转染效率可达90%以上，实验组转染效率达85%以上。RT-PCR和Western blot检测结果显示, 环氧化酶2基因在人骨髓间充质干细胞中的基因水平和蛋白水平均得到了明显抑制。环氧化酶2基因转染对人骨髓间充质干细胞成脂、成骨、成软骨能力无明显影响。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：关键词：骨髓干细胞；人骨髓间充质干细胞；重组慢病毒；环氧化酶2基因；基因沉默；成脂分化；成骨分化；成软骨分化；国家自然科学基金； Abstract：BACKGROUND: At present, research on cyclooxygenase 2 silencing in human bone marrow mesenchymal stem cells is at preliminary stage.OBJECTIVE: To construct the lentivirus vector, and then to observe the expression and effects of cyclooxygenase 2 silencing in the oriented differentiation of human bone marrow mesenchymal stem cells.METHODS: In the present study, the recombinant lentiviral vectors carrying silencing of cyclooxygenase 2 gene and green fluorescent protein were constructed with recombinant lentiviral technology, and then we knocked down cyclooxygenase 2 expression in human bone marrow mesenchymal stem cells through lentivirus infection in vitro (Lenti-ShCOX-2 group). The human bone marrow mesenchymal stem cells transfected with single lentivirals served as Lenti-Shcontrol group. The human bone marrow mesenchymal stem cells with no treatment served as blank control group. At 7 days after trasfection, the total RNA and protein were extracted from eachgroup for detection. The adipogenic, osteogenic, chondrogenic differentiation capacities of these cells were assessed in vitro.RESULTS AND CONCLUSION:After transfected with lentivirus vector for 3 days, we observed the green fluorescence from human bone marrow mesenchymal stem cells with cyclooxygenase 2 silencing under the fluorescence microscope, and the transfection efficiency was over 90% in the Lenti-Shcontrol-2 group and over 85% in Lenti-ShCOX-2 group. We confirmed the successful down-regulation of cyclooxygenase 2 at the mRNA and protein levels in human bone marrow mesenchymal stem cells through lentivirus infection. These findings indicate that cyclooxygenase 2 infection cannot inhibit the differentiation capacity of human bone marrow mesenchymal stem cells.中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：Key words：stem cells;mesenchymal stem cells;cyclooxygenase 2;genes;收稿日期:2014-02-22 中图分类号:R394.2基金资助:国家自然科学基金资助项目(81171774, 81272056)通讯作者:王英振，硕士，博士生导师，青岛大学医学院附属医院关节外科，山东省青岛市 266003 作者简介: 赫天，男，1985年生，黑龙江省哈尔滨市人，满族，2014年青岛大学毕业，硕士，主要从事骨外科的基础与临床研究。引用本文:赫 天,张海宁,项金瑜等. 环氧化酶2基因沉默不影响人骨髓间充质干细胞定向分化的能力J.中国组织工程研究, 2014, 18(14): 2140-2146.He Tian,Zhang Hai-ning,Xiang Jin-yu et al. Cyclooxygenase 2 silencing has no effects on oriented differentiation of human bone marrow mesenchymal stem cellsJ. Chinese Journal of Ti