miR-181a对骨质疏松小鼠骨髓贴壁基质细胞体外增殖活性的影响

Effect of miR-181a on proliferation of bone marrow mesenchymal stem cells from mouse osteoporosis model

目的探讨骨质疏松发生过程中miR-181a对骨髓贴壁基质细胞(mesenchymal stem cells,MSCs)增殖能力的影响。方法选用8周龄健康的20只雌性小鼠,并进行双侧卵巢切除(OVX组),建立雌性小鼠绝经后的骨质疏松模型。另选用同一周龄,体质量相近的20只健康小鼠行双侧卵巢附近脂肪组织部分切除,建立假手术组(Sham组)。2个月后Micro-CT检测2组小鼠松质骨中骨小梁微观结构参数及骨密度指标,MTT检测2组细胞增殖能力的差异。RT-PCR检测2组细胞中miR-181a的表达差异,上调(下调)细胞内miR-181a水平后MTT检测骨髓贴壁基质细胞增殖情况。结果 MicroCT显示Sham组与OVX组骨小梁微观结构参数及骨密度有显著差异(P<0.05)。MTT显示Sham组MSCs增殖能力高于OVX组(P<0.05)。RT-PCR显示Sham组中miR-181a较OVX组表达显著降低(P<0.05)。上调(下调)miR-181a后,MSCs增殖能力下降(上升)。成骨成脂能力检测发现OVX组MSCs成脂能力强于Sham组,而其成骨能力弱于Sham组(P<0.05)。结论雌激素缺乏所导致的骨质疏松症中,miR-181a高表达可抑制MSCs的增殖,可能是影响骨质疏松发病的重要因素之一。

Objective To determine the effect of miR-181 a on the proliferation of mouse bone mesenchymal stem cells( MSCs) derived from mouse model of osteoporosis( OP). Methods Animal model of OP was established by bilateral ovariectomy( OVX) in 20 8-week-old healthy female C57 BL /6 mice,and the mice undergoing sham operation served as control. In 2 months later,micro-CT scanning was employed to detect the parameters of trabecular microstructure and the indicators of the bone mineral density of both groups. The mice were then sacrificed to isolate MSCs,which were then identified by flow cytometry for their phenotypes and by Alizarin red staining and Oil red 0 staining for osteogenic and adipogenic capacities. MTT assay was used to measure the proliferation capacity of the MSCs derived from 2 groups of mice. The mRNA expression of miR-181 a was measured by RT-PCR. The proliferation of MSCs after up-regulating/downregulating the expression of miR-181 a was detected by MTT assay. Results The results of micro-CT scanning showed significant differences in the parameters of trabecular microstructure and the indicators of the bone mineral density between 2 groups( P < 0. 05). The proliferation capacity of MSCs was declined,and the expression of miR-181 a was significantly increased in the cells from OVX group( P < 0. 05). Up-regulation /down-regulation of miR-181 a resulted in significantly decreased/increased proliferation in MSCs. And the adipogenic capacity of MSCs was stronger in OVX group than in sham group,whereas the osteogenic capacity was weaker( P < 0. 05). MTT assay showed the proliferation capacity was better in the MSCs from sham operation group than from OVX group( P < 0. 05). Conclusion In the process of osteoporosis induced by estrogen deficiency,the high expression of miR-181 a inhibits the proliferation of MSCs,which may play an important role in the pathogenesis of postmenopausal osteoporosis.