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背景:基于干细胞的组织工程技术作为治疗骨关节损伤修复的有效手段,具有广泛的应用前景。研究表明miRNA在调控干细胞向软骨分化过程中具有重要的作用。目的:探讨共感染慢病毒介导的反义miR-221-3p/222-3p(microRNA-221-3p,microRNA-222-3p)对人骨髓间充质干细胞成软骨分化的作用,为临床软骨损伤修复提供新的策略。方法:利用miRNA基因芯片技术筛选转化生长因子β3诱导人骨髓间充质干细胞成软骨分化过程中不同阶段表达差异的miRNA,并利用荧光实时定量PCR(RT-q PCR)验证;构建慢病毒人反义miR-221-3p/222-3p载体(Lv-miR-221-3p-inhibition,Lv-miR-222-3p-inhibition)并共转染人骨髓间充质干细胞,空载体组(Lv-GFP)以及未转染组作为对照。利用CCK-8法检测沉默miR-221-3p/222-3p 6 d后细胞的增殖情况;通过番红O染色法、免疫组织化学方法以及RT-PCR验证各组软骨诱导21 d后软骨分化相关标志物的表达。结果与结论:构建的Lv-miRNA-221-3p/222-3p inhibition共转染人骨髓间充质干细胞,成功沉默了细胞中的miR-221-3p/222-3p表达水平;miRNA基因芯片与RT-q PCR验证结果显示miR-221-3p/222-3p在软骨分化后期表达明显降低;转染组与未转染组以及空载体组相比:1细胞增殖明显受到抑制。2番红O染色以及免疫组织化学显示软骨分化特征标志物硫酸软骨素以及Ⅱ型胶原表达增强。3RT-qPCR也证实硫酸软骨素以及Ⅱ型胶原的mRNA表达也明显上调。结果显示沉默人骨髓间充质干细胞miRNA-221-3p/222-3p,抑制了细胞增殖并促进了成软骨分化。
BACKGROUND: Stem cell-based tissue engineering is an effective method for the treatment of bone and joint injuries. It has broad application prospects. Studies have shown that miRNA plays an important role in the regulation of stem cells to differentiate into cartilage. OBJECTIVE: To investigate the effect of lentivirus-mediated antisense miR-221-3p / 222-3p (microRNA-221-3p, microRNA-222-3p) on chondrogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) Damage repair provides a new strategy. Methods: miRNA microarray was used to screen differentially expressed miRNAs induced by transforming growth factor-β3 in human chondrogenic bone marrow mesenchymal stem cells at different stages and confirmed by real-time quantitative PCR (RT-qPCR) MiR-221-3p / 222-3p vector (Lv-miR-221-3p-inhibition, Lv-miR-222-3p-inhibition) and cotransfected into human bone marrow mesenchymal stem cells, empty vector group ) And untransfected group served as control. The proliferation of miR-221-3p / 222-3p cells was detected by CCK-8 method. The cartilage differentiation was detected after 21 days of cartilage induction by Safranin O staining, immunohistochemistry and RT-PCR Marker expression. RESULTS AND CONCLUSION: The constructed Lv-miRNA-221-3p / 222-3p inhibition cotransfected human bone marrow mesenchymal stem cells successfully silenced the expression level of miR-221-3p / 222-3p in cells; miRNA microarray and The results of RT-q PCR showed that the expression of miR-221-3p / 222-3p was significantly decreased in the late stage of cartilage differentiation. Compared with the untransfected group and empty vector group, the proliferation of 1 cell was obviously inhibited. 2 safranin O staining and immunohistochemistry showed cartilage differentiation markers chondroitin sulfate and type II collagen expression increased. 3RT-qPCR also confirmed that mRNA expression of chondroitin sulfate and type II collagen was significantly up-regulated. The results showed that silencing of human bone marrow mesenchymal stem cells miRNA-221-3p / 222-3p inhibited cell proliferation and promoted cartilage differentiation.