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

doi:10.3969/j.issn.2095-4344.2014.14.002

Abstract

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 each group 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

CLC Number:

R394.2

He Tian, Zhang Hai-ning, Xiang Jin-yu, Liu Xiang-ping, Deng Ji-hong, Wang Ying-zhen. Cyclooxygenase 2 silencing has no effects on oriented differentiation of human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(14): 2140-2146.

Figures/Tables 3

48 h以后贴壁细胞开始增大，呈成纤维样，平行排列或漩涡状生长，逐渐形成散在独立的集落，5 d左右即可形成明显集落，七八天可铺满瓶底。传代1 d后细胞贴壁生长，细胞为梭形，类似成纤维细胞，旋涡状或放射状生长，平均两三天可传1代。传代3次后，细胞基本达到纯化，形态均一，无杂质，可用于后续实验。 2.2 环氧化酶2基因shRNA慢病毒载体的鉴定及其滴度环氧化酶2基因shRNA的Oligo DNA经退火形成3对双链DNA，经HpaⅠ和XhoⅠ酶切、pFU-GW-siRNA载体连接、转化后，行PCR鉴定，挑选阳性克隆送测序，结果与预期一致，表明合成的环氧化酶2 shRNA Oligo DNA 序列插入正确。根据各孔的荧光表达量，滴度为1×108 TU/mL。 2.3 慢病毒载体介导的环氧化酶2基因的转染效率每孔细胞随机取3个200倍视野对所有细胞和绿色荧光蛋白阳性细胞计数，计算绿色荧光蛋白阳性细胞百分率，每个感染复数的转染效率为3个视野的平均绿色荧光蛋白阳性细胞百分率。当阴性对照组、实验组感染复数为50时，阴性对照组转染效率均可达90%，实验组转染效率均可达85%(图2)。慢病毒转染后72 h绿色荧光达到最亮，此后保持恒定，光镜下细胞饱满、立体感增加，出现较明显的病毒效应，少部分细胞漂浮、凋亡，贴壁细胞胞质内颗粒增多。 2.4 慢病毒转染后人骨髓间充质干细胞环氧化酶2基因的表达于转染后7 d提取各组细胞的总RNA，进行RT-PCR检测，结果证明相比于阴性对照组和空白组，实验组环氧化酶2基因在人骨髓间充质干细胞中低水平表达(P < 0.05)，而空白组和阴性对照组环氧化酶2基因表达量差异无显著性意义(P > 0.05)，这说明环氧化酶2基因在实验组人骨髓间充质干细胞中成功敲除(图3A)。 2.5 慢病毒转染后人骨髓间充质干细胞环氧化酶2蛋白的表达转染后7 d行Western blot检测，在相同的实验条件下，各组均检测到目的蛋白的表达，实验组的吸光度明显低于空白组和阴性对照组(P < 0.05)，结果提示，与RT-PCR的结果相似，实验组中环氧化酶2蛋白表达量明显减低(图3B，C)。实验证明相比于正常细胞(空白组)而言，单纯的慢病毒载体(阴性对照组)转染对人骨髓间充质干细胞内环氧化酶2的蛋白表达无影响(P > 0.05)。 2.6 人骨髓间充质干细胞慢病毒转然前后成骨、成脂细胞诱导分化后形态学变化成脂细胞诱导分化培养3 d后，细胞内有少量脂滴出现并随细胞排列方向延伸，约第10天脂质增加并融合，细胞逐渐变为圆形，2周左右可见大量脂质沉积，油红O染色呈红色。成骨诱导第3周后胞质内充满颗粒，细胞呈集落样生长，细胞内可见钙质沉积，随后大量钙结节形成，经茜素红染色呈棕红色结节。成软骨诱导培养3周后，甲苯胺蓝染色显示人骨髓间充质干细胞胞内基质均匀蓝染，表明诱导后细胞产生了软骨细胞基质糖胺聚糖。"

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