Polypyrimidine tract binding protein regulates the osteogenic differentiation of bone marrow mesenchymal stem cells
10.3760/cma.j.cn114453-20200330-00191
- VernacularTitle:多聚嘧啶序列结合蛋白在骨髓间充质干细胞成骨分化过程中的作用
- Author:
Zhigang YANG
1
;
Ping DONG
;
Rui CAO
;
Xin FU
;
Xiaoyan LYU
;
Ran XIAO
Author Information
1. 中国医学科学院北京协和医学院整形外科医院研究中心 100144
- Keywords:
Mesenchymal stem cells;
RNA binding proteins;
Polypyrimidine tract bingding protein;
Osteogenesis;
Reactive oxygen species
- From:
Chinese Journal of Plastic Surgery
2020;36(5):551-559
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To clarify the role of polypyrimidine tract binding protein (PTB) in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism.Methods:The bone marrow blood of 4 patients with alveolar cleft treated in Plastic Surgery Hospital of Chinese Academy of Medical Science was collected. Human BMSCs were cultured and induced to osteoblasts, RT-PCR and Western blotting were applied to detect the expression of PTB at different time points. PTB expression in BMSCs was knocked down using shRNA, alizarin red S staining and real-time PCR were applied to evaluate its effect on osteogenic differentiation. Human BMSCs were also treated in vitro with TNF-α, an inflammatory factor closely related to osteoporosis, and PTB expression and osteogenic differentiation were detected by Real-time PCR and alizarin red S staining. The rat osteoporosis model was established and the expression of PTB in the BMSCs was detected by Real-time PCR. Flow cytometry was used to detect intracellular ROS in human BMSCs after PTB knockdown. All quantitative data were displayed as mean ± standard deviation, student t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05. Results:PTB expression increased during osteogenic differentiation of BMSCs in vitro. Compared with the control, the fold changes of relative expressions of osteogenic gene RUNX2 and ALP in BMSCs with PTB knockdown were 0.74±0.15 and 0.29±0.18, respectively, and the decreases were statistically significant( t=3.490, P=0.039; t=7.983, P=0.004). Consist with that, the formation of mineralized nodules decreased after PTB was knocked down. TNF-α at both 10 ng/ml ( t=3.528, P=0.039) and 40 ng/ml ( t=4.306, P=0.023) inhibited PTB expression and osteogenic differentiation of human BMSCs. The relative expression of PTB in BMSCs from ovariectomized rat osteoporosis models (0.001 25±0.000 12) was significantly lower than the sham-operated group(0.001 66±0.000 18)( t=3.217, P=0.032). Compared with the control group (1204.0±300.1), ROS content in BMSCs with PTB knockdown (720.7±129.7) significantly decreased ( t=4.872, P=0.040). Conclusions:PTB promotes the osteogenic differentiation of BMSCs, and PTB knockdown mediated down-regulation of ROS content is an important mechanism for its regulation of osteogenic differentiation of BMSCs.
