The Effect Of Rapamycin On The Aging Model Of Human Maxillofacial Fibroblasts Of Different Ages

Objective:1.Explore the optimal concentration and time of anti-aging of rapamycin(RAPA)for culturing human maxillofacial skin fibroblasts(HSF)in vitro.2.Explore the effects and differences of RAPA on the aging of human maxillofacial skin fibroblasts(HSF)of different ages.Methods:Select the facial skin tissues of adult females discarded from oral and maxillofacial surgery of Suining Central Hospital in 2019-2020:(1)Primary culture of HSF with tissue block adherence method.(2)Observation of cell morphology after HE staining.(3)Immunofluorescence identification,vimentin and keratin identification of fibroblasts.(4)CCK-8 method was used to determine the proliferation ability of HSF after different concentrations of RAPA for different time,and initially screen the optimal concentration and time of RAPA.(5)Aging-related β-galactosidase staining method(SA-β-Gal)further screens the optimal concentration and time.(6)The HSF of different ages will be cultured in groups: the low-age group,the low-age group+R group,the middle-aged group,the middle-aged group+R group,the high-age group,the high-age group+R group:①The SA-β-Gal staining method was used to detect cell senescence.②Use flow cytometry to detect cell apoptosis.③Use qPCR to detect the mRNA expression levels of aging-related factors P16,MMP1,MMP3,COL-1,and COL-3.(7)All results of this experiment were analyzed using Graph Pad Prism 8.0 statistical software.The results are expressed as the mean ± standard deviation.The comparison between the two groups uses independent sample t-test,and the comparison between multiple groups uses one-way analysis of variance;P<0.05 indicates that the difference is statistically significant.Results:The tissue block adheres to the cell culture.It can be seen that there are cells crawling out around the tissue block,and the cells gradually expand into a spindle,polygonal or irregular shape,showing a colony-like growth.HE staining shows that the cells are slender and smooth,long and fusiform,the nucleus is oval and stained with blue,and the cytoplasm is stained with red.Immunofluorescence identification results showed that vimentin was identified as positive and keratin was identified as negative.The results of CCK-8 showed that 0.1nmol/LRAPA treated cell activity for 48h or 72h,and did not inhibit cell proliferation.After SA-β-Gal staining,it can be seen that the cells are slender and long spindle-shaped,and some of the nucleoli are stained blue.Data results show that48h(0.1nmol/L),72h(1nmol/L,10nmol/L),96h(0.5nmol/L,1nmol/L,10nmol/L)compared with the blank control group,SA-β-Gal is positive The staining rate has decreased,and the difference is statistically significant,CCK-8 combined with SA-β-Gal staining to select the optimal concentration and time of RAPA acting on HSF is 0.1nmol/L,48h.The results of SA-β-Gal staining showed that the SA-β-Gal positive staining rate of the low,middle and old HSF experimental group was lower than that of the control group.The results of flow cytometry showed that the ratio of apoptotic cells in the low,middle and old age groups in the control group gradually decreased,and the ratio of viable cells gradually increased.In the experimental group,the ratio of viable cells in the low-and middle-age experimental group was higher than that of the control group,and the ratio of viable cells in the old-age experimental group was lower than that of the control group.Compared with the control group,the ratio of apoptotic cells and necrotic cells in the experimental groups of low,middle and old age decreased.The results of q PCR showed that the relative expression of P16 factor m RNA in the low,middle,and senior age groups after RAPA0.1nmol/L was treated for 48 hours was lower than that of the control group.The relative expression of MMP1 and MMP3 factor m RNA was lower than that of the control group.The relative expression of COL-1 and COL-3 factor m RNA was higher than that of the control group.COL-3 decreased slightly in the middle-aged group after adding the medicine,and the difference was not statistically significant.Conclusion:1.In this study,the optimal concentration of RAPA for HSF anti-aging in vitro is 0.1nmol/L,and the optimal effect time is 48h.2.RAPA has a certain anti-aging effect on HSF of people of different ages,and has a certain difference in the effect of HSF at different ages.Among them,the effect of the middle-age group is more significant,but the follow-up needs to be divided into groups and further research on the age of the middle-age group.