Variation of matrix metalloproteinase 2 levels during Kartogenin-induced directional differentiation of human bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.50.004

Abstract

Abstract:

BACKGROUND: Kartogenin can induce chondrogenic differentiation of mesenchymal stem cells as reported in in vitro experiments. The discovery of Kartogenin finds a novel path to cartilage repair, and it is expected to develop into a new drug to treat osteoarthritis.
OBJECTIVE: To observe the inductive role of Kartogenin in the process of human bone marrow mesenchymal stem cells differentiating into chondrocytes in vitro.
METHODS: In vitro cultured human bone marrow mesenchymal stem cells were grown to the logarithmic phase, and then divided into control group (0 μmol/L Kartogenin), 1 μmol/L Kartogenin group, and 10 μmol/L Kartogenin group. After 72 hours of culture, cell proliferation and differentiation were observed microscopically. Matrix metalloproteinase 2 and type II collagen levels in the cell supernatant were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively.
RESULTS AND CONCLUSION: Under the microscope, Kartogenin was shown to significantly promote the proliferation and differentiation of human bone marrow mesenchymal stem cells. With the increase of Kartogenin concentrations, the level of type II collagen was increased, while the level of matrix metalloproteinase 2 was decreased. These findings indicate that Kartogenin can induce human bone marrow mesenchymal stem cells to differentiate into chondrocytes, and with the increase of Kartogenin concentration, destruction of the cartilage extracellular matrix may be inhibited.

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: Stem Cells, Matrix Metalloproteinase 2, Collagen Type II

CLC Number:

R394.2

Wang Cheng, Bo Qi-yu, Dai Guo-feng, Yang Wei-wei. Variation of matrix metalloproteinase 2 levels during Kartogenin-induced directional differentiation of human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(50): 7475-7480.

Figures/Tables 1

2.1 细胞的形态学观察倒置显微镜下可见大部分细胞呈梭形或不规则形贴壁生长，随着药物浓度的增加，细胞生长更加旺盛，融合增加，呈鱼群样聚集成集落(图1)。空白对照组细胞伸展较好，增殖较慢，排列于皿底呈铺路石状，细胞呈三角形、多角形或梭形，有明显突起，立体感较强。随着药物浓度的逐渐增加，细胞数目逐渐增加，排列紧密，形态逐渐变为细长，胞质丰富，细胞间隙愈加紧密，部分区域重叠生长形成细胞簇。组间差异比较明显。 2.2 MMP-2质量浓度的检测 Elisa法检测各组样本中 MMP-2的吸光度A值，结果见图2。1 μmol/L KGN组(B组)和10 μmol/L KGN组(C组)软骨细胞培养上清中MMP-2水平较空白对照组(A组)明显升高(P=0.001，0.047)，C组与B组比较MMP-2水平明显降低(P=0.016)，统计学结果表明KGN浓度升高可明显降低样本内软骨细胞MMP-2表达。根据Elisa法检测各组样本中MMP-2的吸光度A值，经标准曲线换算为MMP-2的质量浓度值，检测结果显示随着KGN药物浓度增加，MMP-2的表达也随之降低(P=0.03)，见表2。 2.3 Ⅱ型胶原蛋白荧光染色结果免疫荧光染色结果显示，随着KGN浓度的增加，KGN能够明显促进Ⅱ型胶原蛋白的表达(图3)，细胞数目亦随着KGN浓度增加而递增，呈短梭形或多边形，细胞膜及胞浆呈蓝色荧光，细胞核未见明显显示。荧光染色结果可知，KGN能够明显促进骨髓间充质干细胞向软骨细胞分化和Ⅱ型胶原蛋白的表达，药物浓度为10 μmol/L时较1 μmol/L时作用更加显著，通过检测蛋白MMP-2的结果显示，随着KGN药物浓度增加，MMP-2的表达也随之降低，这一结果提示随着诱导药物浓度的增加，软骨细胞外基质的破坏水平也可能得到进一步抑制。"

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