Abstract:

Objective To discuss the effect of 20 （S） - protopanaxadiol （PPD） on the osteogensis differentiation of bone marrow mesenchymal stem cells （BMSCs） of the rats， and to clarify the osteogenesis induction mechanism. Methods The BMSCs were extracted from the SD rats by whole bone marrow method，and the BMSCs were divided into control group and 2.5， 5.0， 10.0， 20.0， and 40.0 mg·L^－1 PPD groups； CCK-8 assay was used to detect the proliferation activities of BMSCs in various groups；the survival of BMSCs in various groups was observed by Calcein/PI staining；the suitable concentration of PPD for the subsequent experiments was selected. The BMSCs were divided into control group and PPD group. On the 7th day of osteogenesis induction， BCIP/NBT colorimetry was used to carry out alkaline phosphatase （ALP） staining and quantitatively detect the activities of ALP in the BMSCs in various groups； On the 21th day of osteogenesis induction， the formations of calcium nodules in the BMSCs in various groups were detected by Alizarin red staining and the mineralization activities of cells in various groups were quantitatively detected； On the 7th day of osteogenesis induction， real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of ALP runt-related transcription factor 2 （Runx-2）， type Ⅰ collagen（COL-1），and osteocalcin （OCN） mRNA in cells in various groups； the immunofluorescence intensities of Runx-2 and OCN protein expressions in the BMSCs in various groups were measured by immunofluorescence staining. Results On the 1st and 3rd days of PPD treatment， compared with control group， the proliferation activity of the BMSCs in 40.0 mg·L^－1PPD group was significantly decreased （P<0.01）； on the 7th day of PPD treatment， compared with control group， there were no significant differences in the proliferation activities of the cells between 2.5，5.0， and 10.0 mg·L^－1 PPD groups （P>0.05），the proliferation activities of the BMSCs in 20.0 and 40.0 mg·L^－1 PPD groups were significantly decreased （P<0.01）. On the 1st and 3rd days of osteogensis induction，the numbers of alive cells in 5.0，10.0，and 20.0 mg·L^－1 PPD groups were more，so 10.0 mg·L^－1 PPD was selected to use for the following experiment.On the 7th day of osteogenesis induction，compared with control group， the activity of ALP in the cells in PPD group was significantly increased （P<0.01）； on the 21st day of osteogenesis induction， compared with control group， the number of mineralized nodules in the cells in PPD group was significantly increased，and the minealized activity was increased （P<0.01）； on the 7th day of osteogenesis induction， compared with control group， the expression levels of ALP，COL-1，OCN，and Runx-2 mRNA in the cells in PPD group were significantly increased（P<0.05），the fluorescence intensities of expressions of Runx-2 and OCN proteins in the cells in PPD group were significantly increased （P<0.01）. Conclusion PPD can promote the osteogenic induction of the BMSCs by increasing the ALP activity and calcium salt deposition in the BMSCs and increasing the expressions of osteogenic genes such as ALP，COL-1，OCN and Runx-2，and PPD is an effective osteogenesis induction activity factor.

Key words: 20（S）-protopanaxadiol, Osteogenesis, Bone marrow mesenchymal stem cells, Cell differentiation, Alkaline phosphatase, Immunofluorescence staining