Effect Of Autophagy On Osteogenic Differentiation Of Maxillary Sinus Membrane Cells

Objective 1.To determine the stem cell characteristics of maxillary sinus membrane cells(MSMCs).2.To establish the model of rapamycin inducing autophagy in MSMCs.3.To explore the effect of autophagy on osteogenic differentiation of MSMCs.Method 1.BMSCs were isolated and purified using whole bone marrow adherent method;MSMCs were isolated and cultured using modified enzyme digestion;flow cytometry(FCM)were performed to identify the phenotype of P3 MSMCs and BMSCs;the ability of MSMCs cloning was detected by crystal violet staining,WST-8 assay compared with BMSCs;osteogenic differentiation was tested by alizarin red staining(ARS)to compare the difference of MSMCs and BMSCs;adipogenic differentiation was tested by oil red O staining to compare the difference of MSMCs and BMSCs;chondrogenic differentiation was tested by oil alcian blue staining to compare the difference of MSMCs and BMSCs.2.Different concentrations of RAPA were used to establish a MSMCs autophagic model;MSMCs forming autophagolysosomes were observed by transmission wlectron microscopy(TEM);monodansylcadaverine(MDC)staining was used to detect the autophagic level of MSMCs qualitatively;FCM was used to detect the autophagic level of MSMCs quantitatively.3.5-bromo-4-chloro-3-indolyl-phosphate/four azole nitro blue(BCIP/NBT)staining was performed to detect the expression of alkaline phosphatase(ALP)in MSMCs qualitatively;p-nitrophenyl phosphate disodium(p NPP)was performed to detect the activity of alkaline phosphatase(ALP)in MSMCs quantitatively;ARS was performed to detect the calcium deposits produced by MSMCs;western blot(WB) was performed to test osteogenesis-related proteins altering with autophagy-related proteins in MSMCs;immunofluorescence(IF)was performed to test test osteogenesis-related proteins mapping dynamically with autophagy-related proteins in MSMCs;reverse transcription-quantitative real time polymerase chain reaction(RT-q PCR)was performed to test osteogenesis-related genes and autophagy-related genes(ATGs)in MSMCs.4.The ectopic bone formation model of different autophagic levels in MSMCs were established;hematoxylin-eosin(HE),masson staining,goldner staining were performed to detect ectopic bone formation qualitatively.Result 1.BMSCs and MSMCs were isolated and cultured successfully;the expression of CD90 and CD146 in MSMCs were positive while the expression of CD34,CD45,CD105 and CD166 in MSMCs were negative;the expression of CD90,CD105,CD146 and CD166 in BMSCs were positive while the expression of CD34 and CD45 in BMSCs were negative;the cloning efficiency of MSMCs was slightly weaker than BMSCs(p<0.05);the logarithmic growth phase of MSMCs was after 4-7 d inoculation,with cell doubling time no weaker than BMSCs(p<0.05);osteogenic differentiation capacity of MSMCs was superior to that of BMSCs;adipogenic differentiation capacity of MSMCs was inferior to that of BMSCs;chondrogenic differentiation capacity of MSMCs was inferior to that of BMSCs.2.After 7 d osteogenic induction,RAPA ranged 0-1000 n M,could induce MSMCs generating gradient increasing autophagolysosomes,with gradient inducing autophagic ratios.3.After 7 d osteogenic induction of MSMCs by RAPA ranged 0-1000 n M,osteogenesis-related proteins and genes(OCN,RUNX2,OPN,COL1)expressions increased first and then dipped,reaching its peak at 100 n M;after 14 d osteogenic induction of MSMCs by RAPA ranged 0-1000 n M,the difference of osteogenesisrelated proteins and genes(OCN,RUNX2,OPN,COL1)expressions decreased;after 21 d osteogenic induction of MSMCs by RAPA ranged 0-1000 n M,the difference of osteogenesis-related proteins and genes(OCN,RUNX2,OPN,COL1) expressions of MSMCs tended to be consistent,while BSP expression increased first and then decreased,reaching its peak at 100 n M;the mapping of osteogenesis-related proteins altered with the autophagic level.Conclusion 1.MSMCs isolated from the maxillary sinus membrane of SD rats could posses the characteristics of high proliferation and multi-directional differentiation of stem cells.The osteogenic potential of MSMCs might be a little stronger than BMSCs.2.RAPA might be effective to induce autophagy in MSMCs,at 0-1000 n M range,while the autophagic level of MSMCs could rise with the concentration of RAPA.3.After 7 d osteogenic induction of MSMCs by RAPA ranged 0-1000 n M,the osteogenic differentiation levels of MSMCs might vary with autophagic levels,increasing first and then decreasing.Over time,after 14 d and 21 d osteogenic induction of MSMCs,the difference of groups of RAPA at different concentrations could tend to be consistent.It indicates that autophagy may plays an important role in the osteogenic differentiation of MSMCs.