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目的应用微小RNAs(microRNAs,miRNAs)芯片技术筛选补肾方含药血清干预大鼠成骨细胞后差异表达的miRNAs,与前期获得的软骨细胞差异miRNAs比较,筛选出共有差异miRNAs,以探讨补肾方通过共有miRNAs调控靶基因表达发挥治疗效应的机制。方法原代培养的大鼠成骨细胞经碱性磷酸酶染色、Ⅰ型胶原免疫荧光染色和矿化结节染色鉴定后,用补肾方含药血清干预成骨细胞,在给定时间点收获细胞,抽提纯化总RNAs行miRNAs芯片检测,并挑选差异表达4倍以上的miRNAs行实时荧光定量逆转录-聚合酶链反应验证。结果所培养的原代细胞具有典型的成骨细胞形态和功能,碱性磷酸酶染色、Ⅰ型胶原免疫荧光染色和矿化结节染色均为阳性。与补肾方含药血清干预成骨细胞1 d相比,干预6 d的共有229个2倍以上表达差异的miRNAs,其中上调的52个、下调的177个。差异表达4倍以上的miRNAs实时荧光定量逆转录-聚合酶链反应结果与芯片数据一致。与前期软骨细胞差异miRNAs芯片数据比较,miRNAs均上调2倍以上的有8个、下调4倍以上的2个。结论补肾方含药血清干预成骨细胞获得了一套较为特异的miRNAs,初步预测上调的miRNAs可能通过靶向Runx2、Wnt、Notch、Axin2等基因在骨发育、骨形态发生、骨吸收以及软骨内成骨形成等相关的信号通路中发挥作用。
OBJECTIVE: To screen miRNAs differentially expressed in rat osteoblasts treated with Bushen Prescription by microRNAs (miRNAs) microarray technology and to compare miRNAs of differentiated chondrocytes obtained in the previous study. A total of miRNAs regulate the expression of target genes play a therapeutic effect mechanism. Methods Primary cultured rat osteoblasts were stained with alkaline phosphatase, type Ⅰ collagen immunofluorescence staining and mineralized nodule staining. Osteoblasts were treated with serum containing Bushen Decoction and cells were harvested at a given time The total RNAs were extracted and purified by miRNA microarray. The miRNAs with more than 4-fold differential expression were selected for real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results The cultured primary cells showed typical morphology and function of osteoblasts. Alkaline phosphatase staining, type Ⅰ collagen immunofluorescence staining and mineralized nodule staining were all positive. Compared with the drug-containing serum from Bushen Recipe for 1 day, there were 229 more than twice as many miRNAs expressed in the 6-day intervention, including 52 up-regulated and 177 down-regulated. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (miRNAs) of miRNAs that differentially expressed more than 4-fold were consistent with the chip data. Compared with the pre-chondrogenic miRNAs, the miRNAs were upregulated more than 2-fold and down-regulated more than 4-fold. CONCLUSIONS: A set of more specific miRNAs were obtained from the intervention of Bushenfang serum on osteoblasts. The miRNAs upregulated initially may be related to bone development, bone morphogenesis, bone resorption and cartilage by targeting Runx2, Wnt, Notch, Axin2 and other genes Osteogenesis and other related signaling pathways play a role.