Effects Of Quercetin On Proliferation And Osteogenic Differentiation Of Rat Gingival Mesenchymal Stem Cells

Objective: SD rat gingival mesenchymal stem cells were cultured and identified,and the effects of quercetin on the proliferation and osteogenic differentiation of rat gingival mesenchymal stem cells were determined by CCK-8 method,alizarin red staining and alkaline phosphatase enzyme linked immunosorbent assay.To investigate the effect of quercetin on rat gingival mesenchymal stem cells in vitro.Methods:1.rGMSCs culture: the maxillary bilateral buccal and palatal gingival tissue of 4-6 weeks old male SD rats were completely removed,and thestem cells were cultured by enzyme digestion and subcultured for 3 to5 generations.2.Identification of rGMSCs: the characteristics of rGMSCs were identifiedby multidifferentiation potential and flow cytometry.3.Detection of cell growth activity: the OD value of rGMSCs at different time(0h,24 h,48h,72 h,96h,120 h,144h,168h)was measured by CCK-8 kit,and the growth curve was drawn.4.Effects of different concentrations of quercetin on the proliferation of rGMSCs: CCK-8 detection kit was used to determine the effect of culture medium containing different concentrations of quercetin(0μM,10μM,20μM,40μM,60μM,80μM)on the proliferation of rGMSCs at different time(0h,24 h,48h,72h).5.Effect of different concentrations of quercetin on osteogenic differentiation of rGMSCs: the content of ALP in osteogenic induction solution containing different concentrations of quercetin(0μM,10μM,20μM,40μM)was determined by ELISA after 7 days.After 21 days,the effect of quercetin on osteogenic differentiation of rGMSCs was observed by alizarin red staining and its integral optical density was analyzed by IPP image and text analysis software.Results:1.rGMSCs can be successfully cultured by enzyme digestion and can differentiate into osteoblasts and lipids under certain induction conditions.Flow cytometry was used to identify the positive expression of mesenchymal stem cell surface markers CD29 and CD90,negative expression of hematopoietic stem cell surface markers CD45.2.The results of CCK-8 assay showed that after the fourth generation cells were inoculated,the latent adaptation period was 1-2 days,the cells increased rapidly after 3 days,and the cells proliferated slowly into the plateau phase after 6 days.3.The results of CCK-8 detection showed that at 24 hours,the OD values of each experimental group(0μM,10μM,20μM,40μM,60μM,80μM)were significantly higher than those of the control group(0μM)and the difference was statistically significant(P<0.05).With the change of time,after24 hours,the OD value of the experimental group(20μM,40μM,60μM,80μM)showed a downward trend.At 72 h,the OD value of the experimental group(40μM,60μM,80μM)was significantly lower than that of the control group,while the OD value of the experimental group(10μM)was significantly higher than that of the control group.The differences were statistically significant(P<0.05).4.The results of ELISA detection showed that different concentrations of quercetin(10μM,20μM,40μM)had different effects on the osteogenic differentiation of rGMSCs.Compared with the control group(0μM),the content of ALP in the experimental group(10μM,40μM)was significantly different(P<0.05).The ALP content of quercetin containing 10μM was significantly higher than that of the control group,while the ALP content of quercetin containing 40μM was significantly lower than that of the control group.There was no significant difference between the experimental group(20μM)and the control group(0μM)(P>0.05).5.The results of alizarin red staining:The inverted microscope show that obvious mineralized nodules were formed in the osteogenic induction solution containing 0μM and 10μM quercetin.The area of mineralized nodules in the osteogenic induction solution containing 10μM quercetin was the largest,while in the osteogenic induction solution containing 20μM and 40μM quercetin,the mineralized nodules were not obvious.The analysis results of IPP graphic analysis software showed that compared with the control group(0μM),the IOD values of the experimental group(10μM,20μM,40μM)were significantly different(P<0.05).The IOD value of quercetin containing 10μM was significantly higher than that of the control group,while the IOD of quercetin containing 20μM and 40μM was significantly lower than that of the control group.Conclusions:1.rGMSCs can be cultured by enzyme digestion and the obtained cells meet the characteristics of stem cell flow identification and osteogenic and adipogenic differentiation.2.The effect of quercetin on the proliferation of rGMSCs was related to the concentration and intervention time,and the effect of quercetin on proliferation was the most obvious when the concentration was 10μM at 72 h.3.The osteogenic induction solution containing 10μM quercetin could promote the osteogenic differentiation of rGMSCs,while 40μM quercetin could inhibit the osteogenic differentiation of rGMSCs.