Runx2 over-expression effect on expression of osteogenesis related genes in osteogenic differentiation of human umbilical cord blood mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.09.024

Abstract

Abstract:

BACKGROUND: Runx2 plays a central role in osteogenic differentiation, which is of important significance in new bone formation.
OBJECTIVE: To observe the effect of Runx2 over-expression on osteogenic differentiation of human umbilical cord blood mesenchymal stem cells.
METHODS: Runx2 was generated by RT-PCR and the recombinant adenovirus (pAd-Runx2) was constructed. The viral titer was determined by air dilution method. After being transfected into human umbilical cord blood mesenchymal stem cells, the green fluorescence was observed under fluorescence microscope. Real-time fluorescent quantitative PCR and western blot were used to detect mRNA expression changes of osteogenesis related genes, Runx2, OCN, BMP-2, ALP, in human umbilical cord blood mesenchymal stem cells at 1, 3, 7 and 14 days after transfection.
RESULTS AND CONCLUSION: The recombinant adenovirus was successfully constructed and the titer was 1.7×10¹⁰ pfu/L. After infection by pAd-Runx2, human umbilical cord blood mesenchymal stem cells expressed green fluorescence protein clearly under the fluorescence microscope. Cells in the transfected group differentiated into osteoblast-like cells, and those in the control group stayed the same as pre-infected. The expression of Runx2, OCN, BMP-2 and ALP in the transfected group increased over time to some extent, but these changes were not detected in the control group. These findings indicate that Runx2 over-expression can promote the osteogenic differentiation of human umbilical cord blood mesenchymal stem cells.

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: Cord Blood Stem Cell Transplantation, Core Binding Factor Alpha 1 Subunit, Adenoviridae, Tissue Engineering

CLC Number:

R394.2

Zhou Da-kai, Li Hui-ning, Ma Shan-shan, Cheng Tian. Runx2 over-expression effect on expression of osteogenesis related genes in osteogenic differentiation of human umbilical cord blood mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1444-1449.

Figures/Tables 2

2.1 重组腺病毒pAd-Runx2的构建提取的RNA具有清晰的18 S和28 S条带，能够满足实验要求。PCR扩增出大小为1 500 bp的特异性条带，测序结果经BLAST比对与GenBank中的Runx2全序列大小一致(图1A)。pAdTrack-CMV-Runx2质粒与pAdEasy1在BJ5183菌体内同源重组后，PacⅠ酶切得到1条约23 kb的片段和1条4 500 bp的片段(图1B)。 2.2 pAd-Runx2的滴度见图2。利用空斑法测定pAd- Runx2的滴度为1.7×1010 pfu/L。 2.3 pAd-Runx2感染脐血间充质干细胞 pAd-Runx2腺病毒进入脐血间充质干细胞并在细胞中成功表达。见图3。 2.4 pAd-Runx2感染脐血间充质干细胞后镜下观察成骨分化情况感染组细胞体积变大，呈鳞片状，细胞核增大。细胞中成骨样细胞数量随培养时间的延长而增加。对照组细胞呈脐血间充质干细胞的典型形态，呈漩涡状生长或呈束生长，形状类似于成纤维细胞，未见成骨样改变(图4)。 2.5 实时荧光定量PCR检测结果感染组Runx2基因的表达量从第3天开始增加，14 d时表达量达到最大值，与对照组比较差异均有显著性意义(P < 0.05，图5A)；感染组OCN基因的表达量从第3天开始增加，14 d时达最大值，但是3，7 d的表达量与对照组比较差异无显著性意义，14 d时明显高于对照组(P < 0.05，图5B)；感染组BMP-2基因的表达量在第1天时无明显变化，3 d时开始增加，7 d时持续增加，与对照组相比差异有显著性意义(P < 0.05)，14 d时达到最大，明显高于对照组(P < 0.05，图5C)；感染组ALP基因的表达量在1，3 d时均无明显变化，7 d时有所增加，14 d时与对照组比较表达量明显增加(P < 0.05，图5D)。 2.6 Western blot检测结果对照组中各时间点成骨相关基因均未见表达，感染组中成骨相关基因随时间推移表达量均有不同程度的上升，其中Runx2和BMP-2表达量的增加最为显著，见图6。"

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