| Objective: To culture mouse osteogenic progenitor cells(MC3T3-E1)from the supernatant of gingival mesenchymal stem cells(GMSCs)co-cultured with T lymphocytes(Jurkat T),observing its effect on the osteogenic properties of MC3T3-E1 cells.To investigate the effect of GMSCs on osteogenesis through the regulation of T lymphocytes in the inflammatory microenvironment to provide the basis for the application of GMSCS in periodontal tissue engineering.Methods: GMSCs were isolated from human gingival tissue and identified.GMSCs were co-cultured with activated Jurkat T lymphocytes in a cell-cell direct contact way,and activated Jurkat T lymphocytes were cultured alone as control.CCK-8 assay was used to detect the proliferation of Jurkat T lymphocytes in the direct contact coculture.The m RNA relative expression levels of pro-inflammatory factors include interleukin-1β(IL-1β),tumor necrosis factor α(TNF-α)and anti-inflammatory factor interleukin-10(IL-10)were detected by q RT-PCR.Supernatant was collected for subsequent experiments.MC3T3-E1 cells were cultured into three groups.Co-culture group(group A)was cultured in medium containing supernatant of co-culture of two kinds of cells;Jurkat T lymphocyte group(group B): culture medium containing supernatant of Jurkat T lymphocyte culture;Control group(group C): the culture medium containing co-culture medium was used.On day 3 and day 7 of culture,the m RNA relative expression levels of collagen type Ⅰ(Col-Ⅰ),alkaline phosphatase(ALP)and Runx-related transcription factor2(Runx2)in MC3T3-E1 cells of the three groups were detected by q RT-PCR.Osteopontin(OPN)and COL-Ⅰ were stained by immunofluorescence.Those experiments were to evaluate the effect on osteogenic properties of MC3T3-E1 cells.Results: GMSCs were successfully isolated and identified by enzymatic digestion and limited dilution methods.CCK-8 results showed that GMSCs could inhibit the proliferation of activated Jurkat T lymphocytes,and this inhibition was positively correlated with cell density.q RT-PCR showed that co-culture with GMSCs could decrease the m RNA expression of IL-1β and TNF-α,and promote the expression of IL-10(P < 0.05).The q RT-PCR results for MC3T3-E1 showed that the m RNA relative expression levels of Col-Ⅰ,ALP and Runx2 in group A were higher than those in group B,but the relative expression levels of the above factors in groups A and B were lower than those in group C(P < 0.05),regardless of the 3d or 7d culture.The results of OPN and COL-Ⅰ immunofluorescence staining showed that in the 3d group,the average fluorescence intensity of COL-Ⅰ in group C was > group A> group B,but there was no statistical significance between group A and B(P > 0.05).In the 7d group,the average fluorescence intensity of COL-Ⅰ was that of group C,>,A,>,B(P < 0.05).The mean fluorescence intensity of OPN showed the same trend on 3d and 7d,which was: group C >group A>group B(P < 0.05).Conclusion: 1.Human gingival mesenchymal stem cells can be successfully isolated from gingival tissue by enzyme digestion and limited dilution method,and the obtained cells have the characteristics of mesenchymal stem cells.2.GMSCs can inhibit the biological activity of activated Jurkat T lymphocytes,down-regulate the expression of pro-inflammatory cytokines m RNA,up-regulate the expression of anti-inflammatory cytokines m RNA,and inhibit the proliferation of activated Jurkat T lymphocytes.3.Gingival mesenchymal stem cells modulate the biological activity of activating Jurkat T lymphocytes to reduce the inhibitory effect on osteoblast differentiation of MC3T3-E1 cells in an inflammatory microenvironment. |
PDF Full Text Request