中国组织工程研究

中国组织工程研究

• 骨髓干细胞 bone marrow stem cells •    下一篇

共沉默miR-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促成软骨分化

闫继红，杨  姝，孙海梅，曹丹丹，张秀英 ,季凤清，郭  多，吴  波，孙婷怡，周德山   

  1. 首都医科大学基础医学院人体解剖与组织胚胎学系组织胚胎学教研室，北京市  100069
  • 出版日期:2015-06-10 发布日期:2015-06-10

Down-regulation of miR-221/222 inhibits cell proliferation and promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells

Yan Ji-hong, Yang Shu, Sun Hai-mei, Cao Dan-dan, Zhang Xiu-ying, Ji Feng-qing, Guo Duo, Wu Bo,
Sun Ting-yi, Zhou De-shan   

  1. Department of Histology & Embryology, Capital Medical University, Beijing 100069, China
  • Online:2015-06-10 Published:2015-06-10

PDF

468

被引次数

7

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，micro-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-qPCR)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-qPCR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。

关键词: 干细胞, miR-221-3p, miR-222-3p, 慢病毒表达载体, 骨髓间充质干细胞, 成软骨分化, 沉默miRNA, 国家自然科学基金

Abstract:

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 miRNAs 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: miRNA microarray technology was applied to detect miRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). 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 mRNA expression at 21 days of chondrogenic induction.
RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cells. miRNA microarray and RT-qPCR 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 Collagen type II 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 group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cells could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cells.

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