New Approach for Differentiation of Bone Marrow Mesenchymal Stem Cells Toward Chondrocyte Cells With Overexpression of MicroRNA-140
New Approach for Differentiation of Bone Marrow Mesenchymal Stem Cells Toward Chondrocyte Cells With Overexpression of MicroRNA-140
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DOI：
10.1097/mat.0000000000000688
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发表时间：
2018-09-01
期刊：
影响因子：
4.2
通讯作者：
Kazemi, Bahram
中科院分区：
文献类型：
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作者：
Mahboudi, Hossein;Soleimani, Masoud;Kazemi, Bahram
间充质干细胞被广泛地刺激转化生长因子β-3（TGF β 3）用于软骨细胞分化。本研究旨在建立一种通过过表达MicroRNA-140（miR-140）诱导人骨髓间充质干细胞向软骨细胞分化的新方法，并与高密度培养体系中TGF-3诱导的方法进行比较。骨髓间充质干细胞来源于人骨髓。我们制备了重组慢病毒载体，并将其用于HEK-293细胞中的重组慢病毒生产。将转导的细胞在单层培养系统中培养，并在第7、14和21天后收获。采用真实的实时荧光定量聚合酶链反应（RT-PCR）检测软骨特异性基因的mRNA水平。为了证实我们的结果，我们还做了免疫细胞化学技术。用重组慢病毒转导骨髓间充质干细胞（BMSCs），表达miR-140。免疫细胞化学方法证实了处理软骨基质基因后BMSC向软骨细胞分化。实时荧光定量PCR结果显示，与未转导的骨髓基质细胞相比，转染miR-140的骨髓基质细胞中II型胶原和聚集蛋白聚糖的基因表达显著增加，I型胶原的表达下调。这些结果与作为阳性对照的TGF-3诱导方法相一致。在这项研究中，我们描述了一种新的方法和技术，可以应用于骨髓基质细胞分化为软骨细胞，而不是刺激与TGF-3。我们的数据表明，miR-140是一种有效的骨髓基质细胞软骨分化诱导剂，我们已经表明，增加软骨分化通过使用miR-140过表达。
Mesenchymal stem cells are widely stimulated by transforming growth factor beta-3 (TGF3) for chondrocyte differentiation. The objective of our study was to establish a new method for differentiation of human mesenchymal stem cells toward chondrocyte by overexpression of MicroRNA-140 (miR-140), and also this method was compared with method of induction with TGF3 in high-cell density culture systems. Mesenchymal stem cells were harvested from bone marrow of human. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were cultured in monolayer culture system and were harvested after days 7, 14, and 21. Real time polymerase chain reaction (RT-PCR) was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry technique. Bone marrow mesenchymal stem cells (BMSCs) were transducted with recombinant Lenti virus, and miR-140 was expressed. Immunocytochemical method confirmed the differentiation of BMSC toward chondrocyte with handling cartilage matrix genes. Also real-time PCR showed that after expression of miR-140 in transducted BMSCs significantly increased gene expression of collagen type II and aggrecan and downregulated expression of collagen type I when compared with the mRNA levels measured in nontransducted BMSCs. These results were compatible compared with TGF3 induction method as control positive. In this study, we described a new approach and technique that may be applied for differentiation of BMSCs to chondrocyte instead of stimulation with TGF3. Our data implies that miR-140 is a potent chondrogenic differentiation inducer for BMSCs, and we have shown increasing chondrogenic differentiation by using miR-140 overexpression.