| Objective: To investigate the effects of macrophage inflammatory protein-1α(MIP-1α)on the proliferation,migration,apoptosis and osteo-differentiation of human periodontalligament stemcells(h PDLSCs)and their process on Notch signaling pathway.Methods: A total of 16 extracted healthy teeth(orthodontic decompensated premolar or interrupted third molar)from November 2020 to October 2021 were collected from patients aged 12 to 25 years old(all signed informed consent)attending the outpatient clinic of the Department of Stomatology,Second Affiliated Hospital of Xinjiang Medical University,and primary stem cells were cultured by tissue block method combined with enzymatic digestion,and cell phenotypes were identified by flow cytometry.(1)Cell proliferation,migration and apoptosis experiments: 3rd generation h PDLSCs were divided into control group(0 μg/m L MIP-1α +culture medium),experimental group(1 μg/m L MIP-1α,10 μg/m L MIP-1α,100 μg/m L MIP-1α + culture medium),and cell proliferation ability was detected by CCK-8 method after 1,3,5 and 7 d of intervention,and 48 h after intervention The c-ell migration ability was detected by Transwell,and apoptosis ability was detected by flow assay after 48 h of intervention.(2)Osteo differentiation and Notch signaling pathway assays: 3rd generation h PDLSCs were divided into control(0),1 and10 μg/ml MIP-1α groups,and osteogenic induction solution was added to them.The mRNA and protein expression of osteogenic genes Runt-related transcription factor 2(Runx2),bone bridge protein(OPN)and transcription factor SP7(Osterix)and Notch1 receptor,Jagged1 ligand and downstream factor Hey1 were detected by qRT-PCR and Western Blot after 7 d of intervention.Results:(i)the flow cytometry identification results of h PDLSCs showed that CD90,CD146 and STRO-1 were positively expressed,97.69%,98.93% and 97.95%,respectively;CD34 and CD45 were negatively expressed,2.18% and 1.08%,respectively;(ii)the CCK-8 results showed that intervention 3,5 and 7 d 10 μg/m L MIP-1α group was significantly higher than that of 0 μg/m L MIP-1α group(P<0.05)and 100 μg/m L MIP-1α grou-p was significantly lower than that of 0 μg/m L MIP-1α group(P<0.05);(iii)Transwell migration results showed that after 48 h,the After 48 h,the number of migrated cells in the 10 μg/m L MIP-1α group was higher than that in the 0 μg/m L MIP-1α group(P<0.000 1),and the number of migrated cells in the 100 μg/m L MIP-1α group was lower than that in the 0 μg/m L MIP-1α group(P<0.001);(iv) the results of apoptosis detection by flow cytometry showed that after 48 h of intervention,the apoptosis rate of h PDLSCs in the 10 μg/m L MIP-1α group was higher than that in the 0 μg/m L MIP-1α group(P<0.001).m L MIP-1α group(P<0.001)and the apoptosis rate of h PDLSCs in the 100 μg/m L MIP-1α group was significantly higher than that in the 0 μg/m L MIP-1α group(P<0.001);(5)The results of alkaline phosphatase staining and semiquantification showed that the activity of 1 and 10 μg/m L MIP-1α groups were lower than that of the 0 μg/mL MIP-1αgroup(P<0.05)The results of alizarin red staining and semiquantification showed that the number of mineralized nodules was lower in 1 and 10 μg/m L MIP-1α groups than in 0 μg/m L MIP-1α group(P<0.05);(6)q RT-PCR and Western Blot res-ults showed that h PDLSCs were downregulated in 10 μg/m L MIP-lα intervention c-ompared with 0 μg/m L MIP-1α group Runx2,Osterix and OPN expression,and th-e differences were statistically significant(P<0.05);Notch signaling pathway-relate d molecules results showed that the expression of Notch1 was reduced in 1 and 10 μg/m L MIP-lα concentration groups compared to 0 μg/m L MIP-1α group,and the differences were statistically significant(P<0.05),and 10 μg/m L MIP-lα concent-ration group,the expression of Jagged1 and Hey1 were all reduced compared with the 0 μg/m L MIP-1α group,and the differences were also statistically significant(P<0.05).Conclusion: MIP-1α promotes the proliferation of hPDLSCs and inhibits the osteodifferentiation of hPDLSCs,probably through the inhibition of Notch signaling pathway. |
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