Effects of oligodeoxynucleotide MT01 on biological characteristics of rat bone marrow mesenchymal stem cells
10.3760/cma.j.cn114453-20190619-00195
- VernacularTitle:寡脱氧核苷酸MT01对大鼠骨髓间充质干细胞生物学特性的影响
- Author:
Yu CHEN
1
;
Pinghui ZHOU
;
Jingjing GUAN
;
Mengxiang LIANG
;
Li ZHANG
;
Yingji MAO
Author Information
1. 蚌埠医学院第一附属医院整形外科 233004；蚌埠医学院生命科学学院 233030
- Keywords:
Oligodeoxynucleotides;
Bone marrow mesenchymal stem cells;
Morphology;
Proliferation;
Osteogenic differentiation
- From:
Chinese Journal of Plastic Surgery
2020;36(5):560-567
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects of oligodeoxynucleotide (ODN) MT01 on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).Methods:The BMSCs of SD rat were isolated and cultured by direct adherence method . The extracted cells were identified by cell morphology of different generations, the expression of surface markers detected by flow cytometry and osteogenic differentiation potential. ODN MT01 group was set up in a gradient of concentrations (0.5, 1.0, 2.0, 4.0 μg/ml) and PBS group as control. Each group of experiments was repeated three times. The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin, respectively. The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1, 4 and 7 d. The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase (ALP) staining, ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium. Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA, respectively. Differences were regarded as statistically significant when a P value of less than 0.05. Results:Flow cytometry showed that the BMSCs were positive for CD29 (99.8%) and CD44 (96.1%) while negative for CD11b (1.03%) and CD45 (1.74%). ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast. The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0 μg/ml. CCK-8 assay showed that the absorbance value of control group was 0.446±0.018, 1.0 μg/ml ODN MT01 was 0.505±0.019, 2.0 μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days. Compared with the control group, the difference is statistically significant ( t=2.954, 4.083, P=0.033, 0.008). The absorbance value of control group was 0.514±0.027, 1.0 μg/ml ODN MT01 was 0.607±0.007, and 2.0 μg/ml ODN MT01 was 0.636±0.023 after cultured for 7 days. Compared with the control group, the difference was statistically significant ( t=4.664, 6.091, P=0.009, 0.008). The proliferation ability of BMSCs was significantly higher than that of the control group. However, 4.0 μg/ml ODN MT01 (0.427±0.013) had an inhibitory effect on the proliferation ability of BMSCs ( t=4.332, P=0.0149). The blue mass and mineralized nodule improved significantly with the increase of ODN MT01 concentration during the induction of osteogenic differentiation of BMSCs. After cultured for 4 days, the result of ALP activity assay was similar to ALP staining. The activity value of ODN MT01 in the control group was 1.207±0.023, 0.5 μg/ml ODN MT01 was 1.747±0.095, 1.0 μg/ml ODN MT01 was 2.200±0.136, 2.0 μg/ml ODN MT01 was 3.560±0.088, 4.0 μg/ml ODN MT01 was 3.490±0.144. Compared with the control group, the difference was statistically significant ( t=4.313, 7.934, 18.800, 18.240; P=0.005, 0.001, <0.001, <0.001). But there was no difference between 2.0 and 4.0 μg/ml groups ( t=0.562, P=0.590). Conclusions:ODN MT01 with concentration of 2.0 μg/ml could significantly stimulate the proliferation and osteogenic differentiation of BMSCs without affecting the morphology of BMSCs.
