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

Cyclooxygenase 2 silencing has no effects on oriented differentiation of human bone marrow mesenchymal stem cells

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。

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 ceils. METHODS： In the present study, the recombinant lentiviral vectors carrying silencing of cyclooxygenase 2 gene and green fluorescent protein were constructed with recombinant lentivirel 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 trensfected 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.