Effects of sclerostin-single chain antibody fragment on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0402

Abstract

Abstract:

BACKGROUND: Antagonism of bone sclerosis protein can stimulate osteogenesis and increase bone synthesis and metabolism through the Wnt/β-catenin signaling pathway.
OBJECTIVE: To investigate the effects of sclerostin-single chain antibody fragment (Scl-scFv) on the proliferation and osteogenic differentiation of C57BL/6 mouse bone marrow mesenchymal stem cells (BMSCs).
METHODS: BMSCs were isolated from C57BL/6 mice using whole bone marrow adherence method. Alizarin red staining was performed at the 14th day of osteogenic induction, and oil red O staining performed at the 7th day of adipogenic induction. Passage 3 BMSCs were cultured with α-MEM complete medium with (experimental) or without (control) 50 μg/L Scl-scFv aScl-scFv. Real-time PCR was used to detect type 1 collagen, alkaline phosphatase, RUNX2, osteopontin, osteocalcin at the 7th day of culture and meanwhile, alkaline phosphatase staining was done; western blot assay was used to detect expression of type 1 collagen and osteopontin proteins, and ELISA was used to detect the level of osteocalcin in the cell supernatant at 4, 7, 10 days of culture.
RESULTS AND CONCLUSION: Formation of calcium nodules and orangered oil droplets was obviously visible in the BMSCs after osteogenic and adipogenic induction, respectively. Over time, the absorbance value showed no difference between the experimental and control group. Compared with the control group, the experimental group showed significant increases in mRNA and protein expression of type 1 collagen and osteopontin as well as in protein expression of alkaline phosphatase, RUNX2 and osteocalcin (P < 0.05). Moreover, stronger alkaline phosphatase staining was found in the experimental group relative to the control group. These findings indicate that Scl-scFv has no effect on BMSCs proliferation, but can promote the osteogenic capacity of BMSCs in vitro.

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: Bone Marrow, Mesenchymal Stem Cells, Single-Chain Antibodies, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Li Shi-fei, Zhu Meng-hai, Zhang Shu-dong, Yao Qi. Effects of sclerostin-single chain antibody fragment on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 1-6.

Figures/Tables 2

2.1 骨髓间充质干细胞形态接种后4 h即可见细胞贴壁生长，呈亮圆形的单核细胞。1 d后首次半量换液去除未贴壁细胞，贴壁细胞胞体明显增大，细胞内颗粒增多，2 d后梭形生长的细胞数量明显增多，偶可见胞体有多个突起的细胞，呈多角形，立体感不强。3 d后全量换液，细胞形态以长梭形为主，可见克隆。4 d后分散生长细胞减少或消失，克隆明显增多，细胞多呈漩涡状或放射状层叠交织生长，为成纤维细胞样特征。7 d左右细胞汇合率达80%，在原代培养的单核细胞中还可见异质细胞，这些异质细胞及圆形的单核细胞均随传代而逐渐减少，细胞形态比较单一，呈漩涡状或放射状平行排列。见图1。 2.2 成脂诱导油红O染色结果实验组和对照组培养7 d后油红O染色，实验组细胞中可见大小不等的圆形红色脂滴，对照组没有脂滴，见图2。 2.3 成骨诱导茜素红染色结果成骨诱导培养和Scl-scFv作用14 d后，可见散在钙结节，实验组茜素红染色强于阳性对照组和对照组，见图3。 2.4 细胞增殖情况对照组的吸光度值与实验组的吸光度值差异无显著性意义，提示Scl-scFv对骨髓间充质干细胞增殖的作用可能不大，见图4。 2.5 Scl-scFv对骨髓间充质干细胞成骨基因表达的影响在Scl-scFv作用下，实验组成骨基因Col1、ALP、RUNX2、OPN、OCN均有一定程度的上调，与对照组比较差异有显著性意义(P < 0.05)，见图5。 2.6 Scl-scFv作用下骨髓间充质干细胞成骨分化过程中碱性磷酸酶的表达骨髓间充质干细胞在成骨分化过程中表达的碱性磷酸酶可以将底物去磷酸化，是评价成骨活性的早期指标。细胞培养7 d后进行碱性磷酸酶染色，碱性磷酸酶表达阳性的部位被染成蓝色，结果发现，实验组细胞染色强度和阳性细胞数目都强于阳性对照组和对照组，见图6。 2.7 Scl-scFv对骨髓间充质干细胞成骨分化过程中相关蛋白检测结果 2.7.1 Western blot检测Col1、OPN蛋白表达见图7。实验组Col1和OPN的表达强于对照组。 2.7.2 ELISA检测骨钙素水平见图8。实验组细胞上清中骨钙素水平较对照组显著升高，差异有显著性意义(P < 0.05)。"

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