目的:探究miR-17-5p对颅缝处颅骨的间充质干细胞(SuSCs)成骨分化的调节作用,并进一步揭示其可能的作用机制。方法:(1)提取并培养SD大鼠SuSCs,使用成骨诱导液诱导后,利用碱性磷酸酶(ALP)染色、茜素红染色、RT-qPCR来鉴定其成骨能力,再利用流式细胞术鉴定所提取SuSCs的纯度。(2)构建miR-17-5p的过表达和干扰载体并转染至SuSCs细胞,利用ALP染色、茜素红染色、RT-qPCR来检测过表达或干扰miR-17-5p对SuSCs成骨分化的影响。(3)过表达或干扰miR-17-5p载体并转染至SuSCs细胞,利用WB检测转染后细胞TGF-β、Smad7的表达。结果:成功提取SuSCs细胞;SuSCs过表达miR-17-5p后ALP染色、茜素红染色、RT-qPCR检测成骨相关标志物(ALP、OCN、Runx2、BMP2)的结果均展现出促进成骨分化的趋势;而SuSCs干扰miR-17-5p后,ALP染色、茜素红染色、RT-qPCR检测成骨相关标志物(ALP、OCN、Runx2、BMP2)的结果均展现出抑制成骨分化的趋势。WB结果显示,相较于空白对照组,过表达miR-17-5p显著提高了TGF-β蛋白的表达水平,同时显著降低了Smad7蛋白的表达水平;而干扰miR-17-5p则展现出相反结果。结论:miR-17-5p参与SuSCs的成骨分化,并通过调节TGF-β/Smad7信号通路发挥关键作用。
Objective: To investigate the regulatory role of miR-17-5p in the osteogenic differentiation of suture mesenchymal stem cells (SuSCs) and further explore its potential mechanism. Methods: (1)SuSCs of the cranial suture in SD rats were extracted and cultured. After induction with osteogenic induction solution, the expression levels of osteogenic markers (ALP, OCN, Runx2, BMP2) were detected by alkaline phosphatase (ALP) staining, alizarin red staining and RT-qPCR, respectively. The osteogenic ability was identified, and the purity of the extracted SuSCs was identified by flow cytometry. (2)The overexpression and interference vectors of miR-17-5p were constructed and transfected into SuSCs cells. The expression levels of osteogenic markers (ALP, OCN, Runx2, BMP2) were detected by alkaline phosphatase (ALP) staining, alizarin red staining and RT-qPCR, respectively. The effects of overexpression or interference of miR-17-5p on osteogenic differentiation of SuSCs were detected. (3)After overexpressing or interfering with miR-17-5p and transfecting SuSCs cells, WB was used to detect the mechanism of its effect on TGF-β and Smad7. Results: SuSCs cells were successfully extracted. The results of ALP staining, alizarin red staining and RT-qPCR detection of osteogenesis-related markers (ALP, OCN, Runx2, BMP2) after overexpression of miR-17-5p in SuSCs showed a trend of promoting osteogenic differentiation. After SuSCs interfered with miR-17-5p, ALP staining, alizarin red staining and RT-qPCR detection of osteogenic markers (ALP, OCN, Runx2, BMP2) showed a trend of inhibiting osteogenic differentiation. WB results showed that compared with the blank control group, overexpression of miR-17-5p significantly increased the expression level of TGF-β protein and significantly decreased the expression level of Smad7 protein, while interfering with miR-17-5p showed the opposite results. Conclusion: miR-17-5p is involved in the osteogenic differentiation of SuSCs and exerts a critical regulatory role by modulating the TGF-β/Smad7 signaling pathway.
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