Study on proliferation effects of low intensity pulsed ultrasound on human bone marrow mesenchymal stem cells in vitro

Abstract

Objective To investigate the effect of low intensity pulsed ultrasound (LIPUS) on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs) and the effect of Cyclin D1 on the proliferation of hBMSCs induced by LIPUS. Methods The cells were inoculated on the a 12-well culture culture plate at a cell concentration of 2.0×104 cells/ml, LIPUS with radiation intensity of 0, 30, 40, 50, 60 and 80 mW/cm2 was used to stimulate for 5 min, the morphological changes of the cells were observed at 0, 24, 72 and 96 h, cell counting was performed with a Scepter 2.0 handheld cell counter, the cell cycle and value-added index were measured by flow cytometry, the cell proliferation was detected by cell counting kit-8 (CCK-8). The expression of Cyclin D1 was detected by Western blotting. The expression of Cyclin D1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Cell counts were found except for the 30 mW/cm2 (P=0.871) group that the number of cells in 40 W/cm2 (P=0.006), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.004) group was significantly higher than that in control group. Cell proliferation assay was found that the proliferation of 30 mW/cm2 (P=0.042), 40 W/cm2 (P=0.012), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.046) cells was significantly higher than that of the control group. Flow cytometry detection found that the cell proliferation index of LIPUS group was significantly higher than that of control group (P=0.001). Western blotting and RT-PCR showed that there was no significant difference between the 30 mW/cm2 group and the control group. The expression of Cyclin D1 was significantly increased in 40 W/cm2 (P=0.002), 50 W/cm2 (P=0.004), 60 W/cm2 (P=0.023), 80 W/cm2 (P=0.012) groups. The expression of Cycling D1 mRNA was significantly different from that of control group (P=0.001). Conclusion LIPUS can significantly promote the proliferation of hBMSCs, and promote the proliferation of hBMSCs from G1 phase to S phase by up-regulation of Cyclin D1 expression. Key words: Low intensity pulsed ultrasound; Human bone marrow mesenchymal stem cells; Proliferation; Cyclin D1