目的 探讨人脐带间充质干细胞(hucMSCs)源性外泌体对成骨细胞增殖和分化的影响及可能的调控机制.方法 培养hucMSCs,采用超速离心法分离hucMSCs源性外泌体(hucMSCs-Exos)并鉴定;设置并对比空白对照组与hucMSCs-Exos共孵育组人成骨细胞hFOB1.19内LINC00520的表达变化;构建抑制或促进LINC00520表达的质粒,分为shLINC00520、LINC00520阴性对照短发卡RNA(shRNA,shNC)、LINC00520 OE和LINC00520 OE的阴性对照Vector 4组,并处理hucMSCs-Exos;检测共孵育后hFOB1.19细胞增殖和分化及L1NC00520的表达水平.采用独立样本t检验进行统计学分析.结果 超速离心法可以有效提取hucMSCs-Exos.hucMSCs-Exos共孵育组hFOB1.19细胞内LINC00520(4.07±0.18)明显高于空白对照组(0.95±0.06),差异有统计学意义(t=-32.89,P＜0.05).LINC00520 OE组胞质内矿化沉淀物明显多于shNC组.LINC00520 OE组成骨分化相关因子 OCN、RUNX2、Collagen I 和 ALP 的 mRNA(1.90±0.13、4.53±0.18、3.80±0.18、1.74±0.08)均高于 Vector 组(0.94±0.16、0.96±0.13、0.96±0.14、0.49±0.10),差异均有统计学意义(t=-9.31、-32.16、-24.91、-19.52,P＜0.01).结论 hucMSCs-Exos 可能通过LINC00520调控成骨细胞增殖和分化.
Human umbilical cord mesenchymal stem cells derived exosomes promote the proliferation and differentiation of hFOB1.19 via LINC00520
Objective To evaluate the effects of human umbilical cord mesenchymal stem cell(hucMSCs)-derived exosomes(hucMSCs-Exos)on osteoblast proliferation and differentiation and possible regulatory mechanisms.Methods The hucMSCs were cultured,and the hucMSCs-derived exosomes(hucMSCs-Exos)were isolated and identified by ultracentrifugation;the expression changes of LINC00520 in human osteoblasts hFOB1.19 were set up and compared between the blank control group and the co-in-cubation group of hucMSCs-Exos;and the plasmids that inhibit or promote the expression of LINC00520 were constructed,and classified into four groups shLINC00520,short hairpin RNA(shRNA)of LINC00520 negative control(shNC),LINC00520 OE,and negative control Vector of LINC00520 OE and treated hucMSCs-Exos respectively;Cell proliferation and differentiation and expression level of LINC00520 in hFOB1.19 cells were detected in each group.Each index was statistically analyzed by independent sam-ple t-test.Results Ultracentrifugation could effectively extract hucMSCs-Exos.hFOB1.19 intracellular LINC00520 in hFOB1.19 cells of hFOB1.19 co-incubation group(4.07±0.18)was significantly higher than that in the blank control group(0.95±0.06),and the difference was statistically significant(t=-32.89,P＜0.05).hFOB1.19 intracellular mineralized precipitates of LINC00520 OE group were signif-icantly higher than that of hNC group.There were significantly more intracytoplasmic mineralized precipi-tates in hFOB1.19 cells than in the shNC group.mRNAs of OCN,RUNX2,Collagen I and ALP were higher in the LINC00520 OE group(1.90±0.13,4.53±0.18,3.80±0.18,1.74±0.08)than in the Vector group(0.94±0.16,0.96±0.13,0.96±0.14,0.49±0.10),and the differences were all statis-tically significant(t=-9.31,-32.16,-24.91,-19.52,P＜0.01).Conclusion HucMSCs-Exos may regulate functions such as osteoblast proliferation and differentiation through LINC00520,which is ex-pected to be a new therapeutic target for the treatment of osteoporosis.
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