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目的:观察微小RNA(microRNA，miR)-219a-5p在骨髓间充质干细胞(BMSC)中的表达及其对细胞增殖和成骨分化的影响。方法:选取在2019年1月至2020年1月期间于河南省人民医院骨科接受髋关节置换的16例激素性股骨头坏死患者及9例股骨颈骨折不愈合患者分别作为实验组及对照组，提取股骨近端BMSC，利用实时定量反转录聚合酶链反应(RT-qPCR)检测miR-219a-5p和卷曲蛋白4(FZD4)在细胞中的表达水平。荧光素酶活性检测验证miR-219a-5p和FZD4的相互作用关系。利用细胞转染技术和miR-219a-5p的模拟物、抑制剂、FZD4发卡RNA(shRNA)来调控BMSC中miR-219a-5p和FZD4的表达水平。细胞计数试剂盒(CCK-8)增殖实验检测BMSC的增殖活性，茜素红染色评估BMSC的成骨分化能力。利用统计与制图软件(GraphPad Prism)统计分析实验数据。采用方差分析(ANOVA)和n t检验确定计量资料各组间差异的统计学意义，计数资料的组间比较采用方差检验。n 结果:miR-219a-5p在实验组BMSC中的相对表达量为6.03±1.39，而在对照细胞中的相对表达水平仅有1.01±0.21，两组之间差异有统计学意义(n t＝10.351，n P<0.01)。利用数据库TargetScan预测得到miR-219a-5p的下游靶基因FZD4，RT-qPCR结果提示FZD4在实验组细胞中的表达水平为1.09±0.55，在对照组细胞中相对表达量达到4.61±1.08，两组之间差异有统计学意义(n t＝10.376，n P<0.01)。皮尔森(Pearson)线性分析显示miR-219a-5p和FZD4在BMSC中表达水平呈负相关[n r＝-0.631，95%可信区间(n CI)：-0.763、-0.498，n P<0.01]。下调实验组中miR-219a-5p的表达后，FZD4的水平升高4.34倍(n t＝8.426，n P<0.01)，而在对照组细胞中上调miR-219a-5p的表达后，FZD4的表达水平下降4.75倍，差异均有统计学意义(n t＝8.667，n P<0.01)。荧光素酶活性检测证实了miR-219a-5p与FZD4之间的结合关系。CCK-8实验结果显示实验组BMSC培养24、48、72 h后的吸光度值分别为0.27±0.05、0.48±0.05、0.63±0.04显著低于对照组的BMSC的0.46±0.05、0.96±0.08、1.51±0.11，两组之间差异具有统计学意义(n t＝9.776，n P<0.01)。抑制实验组中miR-219a-5p表达后细胞的增殖活性明显提高，而同时敲低FZD4的表达则会逆转上述作用。同样地，将miR-219a-5p模拟物转染对照组细胞，细胞增殖活性明显抑制。茜素红染色显示对照组细胞的阳性染色面积/总面积为0.58±0.05，明显高于实验组的0.22±0.04，差异有统计学意义(n t＝7.610，n P<0.01)。在实验组细胞中转染miR-219a-5p抑制剂可以增强细胞的成骨分化能力，但FZD4 shRNA抵消了miR-219a-5p抑制剂对成骨能力的强化作用，且上调正常BMSC中miR-219a-5p的水平可以阻止其成骨分化过程。n 结论:miR-219a-5p在激素性股骨头坏死BMSC中高表达，FZD4介导了miR-219a-5p对BMSC增殖及成骨分化的抑制作用。“,”Objective:To investigate the expression of microRNA (miR)-219a-5p in bone marrow mesenchymal stem cells (BMSCs) and its effect on the proliferation and osteogenic differentiation of BMSCs.Methods:A total of 16 patients with steroid-induced osteonecrosis of the femoral head and 9 patients with nonunion of femoral neck fracture who underwent hip arthroplasty were selected as experimental group and control group respectively. The expression levels of miR-219a-5p and Frizzled-4 (FZD4) in BMSCs were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Luciferase activity assay was used to verify the interaction between miR-219a-5p and FZD4. The expression levels of miR-219a-5p and FZD4 in BMSCs were regulated by cell transfection with miR-219a-5p mimic, inhibitor and FZD4 small hairpin RNA(shRNA). Cell counting kit (CCK-8) was used to detect the proliferation activity of BMSCs. Alizarin red staining was used to evaluate the osteogenic differentiation ability. The experimental data were analyzed by GraphPad Prism software. Analysis of variance and n t-test were used to determine the statistical significance of measurement data among groups, and variance test was used to compare count data among groups.n Results:The relative expression of miR-219a-5p in BMSCs of experimental group was 6.03±1.39, and that in control cell was only 1.01±0.21, with the difference being statistically significant (n t=10.351, n P<0.01). Using the database TargetScan to predict the downstream target of miR-219a-5p, RT-qPCR showed that the expression level of FZD4 in the experimental group was 1.09±0.55, and that in the control cells was 4.61±1.08, with the difference being statistically significant (n t=10.376, n P<0.01). Pearson linear analysis showed that there was a negative correlation between miR-219a-5p and FZD4 in BMSCs [n r=-0.631, 95% confidence interval (n CI): -0.763, -0.498, n P<0.01]. After down-regulating the level of miR-219a-5p in experimental group, FZD4 level increased by 4.34 times (n t=8.426, n P<0.01), and in control group, the expression of FZD4 decreased by 4.75 times after up-regulating miR-219a-5p (n t=8.667, n P<0.01). Further luciferase activity assay confirmed the binding relationship between miR-219a-5p and FZD4. The results of CCK-8 showed that the absorbance values of experimental group were 0.27±0.05, 0.48±0.05 and 0.63±0.04 after 24, 48 and 72 h of culture respectively, which were significantly lower than those of the control group (0.46±0.05, 0.96±0.08, 1.51±0.11, respectively). The proliferation activity of BMSCs in the experimental group was significantly increased after miR-219a-5p was inhibited, and the above effects were reversed by knockdown of FZD4. Besides, miR-219a-5p mimic inhibited cell proliferation in normal BMSCs. Alizarin red staining showed similar results. The positive staining area/total area of control group was 0.58±0.05, which was significantly higher than that of the experimental group (0.22±0.04). In the experimental group, miR-219a-5p inhibitor could enhance the osteogenic differentiation ability, but FZD4 shRNA counteracted this effect, and up-regulating the level of miR-219a-5p in normal BMSCs could prevent the osteogenic differentiation process.n Conclusion:miR-219a-5p is highly expressed in BMSCs of steroid-induced osteonecrosis of the femoral head. FZD4 mediates the inhibitory effect of miR-219a-5p on the proliferation and osteogenic differentiation.