摘要
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： The use of mesenchymal stem cells in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cells are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that micro RNAs play critical roles in chondrogenic differentiation of mesenchymal stem cells. OBJECTIVE： To observe the chondrogenic effect of human bone marrow mesenchymal stem cells transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury. METHODS： mi RNA microarray technology was applied to detect micro RNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified byreal-time fluorescence quantitative PCR（RT-q PCR）. Human bone marrow mesenchymal stem cells were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cell counting kit-8 was used to determine the cell proliferation, the differentiation of bone marrow mesenchymal stem cells towards chondrocytes was verified by type II collagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II collagen and aggrecan m RNA expression at 21 days of chondrogenic induction. RESULTS AND CONCLUSION： The expression of mi R-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cells. micro RNA microarray and RT-q PCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II collagen were significantly increased in the miR-221-3p/222-3p inhibition group and cell proliferation was also inhibited significantly compared with non-transduced cells or transduced with the empty lentiviral vector
出处
《中国组织工程研究》
CAS
北大核心
2015年第50期8056-8061,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金面上项目(81271728)
北京市自然科学基金面上项目(7152023)~~
