Effects of miR-218 and miR-26a on osteogenic differentiation of bone marrow mesenchymal stem cells by polyethyleneimine delivery

doi:10.3969/j.issn.2095-4344.2017.25.005

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are widely used in the field of tissue engineering because of their multi-directional differentiation potential. Micro RNAs play an important role in promoting osteogenic differentiation of BMSCs.
OBJECTIVE: To investigate the effect of miR-218 and miR-26a on the osteogenic differentiation of rat BMSCs, and to provide reference for the study on osteogenic differentiation of BMSCs and the clinical application.
METHODS: The bone marrow of the femur of Wistar rats was extracted and the BMSCs were isolated and cultured to the 3rd generation. MiR-218, miR-26a and polyethyleneimine (PEI) were mixed in a specific ratio to form a miRNA/PEI complex. Meanwhile, a negative control group was established.
RESULTS AND CONCLUSION: (1) Rat BMSCs grew well. (2) The optimal concentration of miRNA mimics was 50 nmol/L by MTT method. (3) The expression of alkaline phosphatase and collagen type I mRNA was significantly increased (P < 0.05). (4) Alkaline phosphatase staining showed that compared with the blank control group and the negative control group, the cytoplasm showed obvious coloring. (5) There were a lot of mineralized nodules shown by alizarin red staining. Therefore, miR-218/PEI complex, miR-26a/PEI complex and miR-218+miR-26a/PEI co-transfection complex could promote the osteogenic differentiation of BMSCs. Under the same conditions, the osteogenesis of miR-26a was slightly better than that of miR-218.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: MicroRNAs, Mesenchymal Stem Cells, Polyethyleneimine, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Sun Xuan, Wei Ze-quan, Bu Wen-huan, Fang Tengjiaozi, Xiluopianduo, Liu Qi-lin, Sun Hong-chen. Effects of miR-218 and miR-26a on osteogenic differentiation of bone marrow mesenchymal stem cells by polyethyleneimine delivery[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3964-3970.

Figures/Tables 2

2.1 骨髓间充质干细胞的培养及形态观察原代骨髓间充质干细胞贴壁后呈短梭形或多角形，可见宽度不均的突起，细胞核大，核仁清晰，细胞大小不一。消化传代至第3代，细胞生长速度加快，形态呈长梭形，排列紧密呈漩涡状，密度达80%-90%(图1)。 2.2 不同浓度miR/PEI复合物转染骨髓间充质干细胞 24 h后对增殖能力影响在浓度10-100 nmol/L时miR-218/PEI组与miR-26a/PEI组细胞增殖率随浓度的增加而有下降趋势，但与空白对照组相比差异无显著性意义(P > 0.05)，而与空白对照组相比，100和200 nmol/L miR-218+miR-26a/PEI共转染组细胞增殖率降低(P < 0.01)，其他浓度miR-218+miR-26a/PEI共转染组中细胞增殖率无明显变化(P > 0.05；图2)。 2.3 RT-PCR检测成骨相关基因碱性磷酸酶和Ⅰ型胶原的mRNA表达水平转染后3，7，14 d时，与空白对照组相比，各组碱性磷酸酶和Ⅰ型胶原mRNA水平均有升高(P < 0.05)，其中miR-218/PEI组和miR-26a/PEI组最明显(图3)。 2.4 碱性磷酸酶染色及定量测定成骨诱导7 d后，与空白对照组相比，阴性对照染色面积接近(P > 0.05)，而各组胞质中均产生了较为明显的着色，其中 50 nmol/L miR-218/PEI组与50 nmol/L miR-26a/PEI组胞浆丰富，染色面积和染色程度明显增强，单一转染组碱性磷酸酶阳性细胞比例稍高于共转染组。定量分析也提示miR-26a/PEI组阳性细胞比例稍高于miR-218/PEI组(P < 0.05)，见图4。 2.5 茜素红染色及定量分析成骨诱导21 d后，茜素红染色结果显示，与空白对照组相比，其他组细胞均不同程度地产生了矿化结节(图5A，B)。其中，miR-26a/PEI组矿化结节密度高于miR-218/PEI组。定量分析结果显示， 50 nmol/L miR-218/PEI组、50 nmol/L miR-26a/PEI组、25 nmol/L miR-218+25 nmol/L miR-26a/PEI组和50 nmol/L miR-218+50 nmol/L miR-26a /PEI组细胞中钙化结节含量明显高于空白对照组(P < 0.01)，其中50 nmol/L miR-26a/PEI组钙化结节含量高于50 nmol/L miR-218/PEI组，提示miR-26a成骨能力优于miR-218 (P < 0.01；图5C)。"

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