| Objective: Autophagy may be involved in the occurrence and development of periodontitis.However,in the microenvironment of diabetes with periodontitis,the autophagy of periodontal ligament stem cells(PDLSCs)and its related mechanisms are not clear.To this end,this experiment uses autophagy as a research entry point to further explore the relationship between diabetes and periodontitis.Methods: In this experiment,periodontal ligament tissues of healthy people,patients with simple diabetes,patients with simple chronic periodontitis,and patients with type 2diabetes with periodontitis were cultured by enzymatic tissue block method,and the cultured cells were cloned by limiting dilution method Cultured and purified into periodontal ligament stem cells.Flow cytometry was used to detect cell-related phenotypic molecules.The autophagy inhibitor 3-methyladenine(3-MA)(10nmol/L)and autophagy activator rapamycin(RAPA)(50ug/L)were used for drug intervention in each group.Experimental grouping(a total of 12 groups):(1)Healthy group(H-PDLSCs group);(2)Healthy + 3-MA group(H-PDLSCs + 3-MA group);(3)Healthy + Rapa group(H-PDLSCs + Rapa group);(4)Simple diabetes group(D-PDLSCs group);(5)Simple diabetes + 3-MA group(D-PDLSCs + 3-MA group);(6)Simple diabetes + Rapa group(D-PDLSCs + Rapa group);(7)Simple periodontitis group(P-PDLSCs group);(8)Simple periodontitis + 3-MA group(P-PDLSCs + 3-MA group);(9)Simple periodontitis+ Rapa group(P-PDLSCs + Rapa group);(10)Diabetes with periodontitis group(DP-PDLSCs group);(11)Diabetes with periodontitis + 3-MA group(DP-PDLSCs + 3-MA group);(12)Diabetes with periodontitis + Rapa group(DP-PDLSCs + Rapa group).The CCK-8 method was used to detect the proliferation of the cells in each of the above groups.Cell autophagy and organelle destruction were observed by transmission electron microscopy.RT-q PCR was used to detect the relative expression of autophagy genes(Beclin-1,LC3Ⅱ,P62)in each group.Results:1.Isolation and culture of periodontal ligament stem cells: 12 cases of H-PDLSCs,3 cases of D-PDLSCs,7 cases of P-PDLSCs,3 cases of DP-PDLSCs were successfully cultured.There were no significant differences in morphology of the four groups of PDLSCs from different sources.They were all spindle-shaped fibroblast-like morphologies.2.Identification of periodontal ligament stem cells: Flow cytometry was used to detect phenotype molecules related to PDLSCs in the 3rd generation of 4 groups.The results are as follows: The surface antigens of H-PDLSCs are: CD90(99.86%),CD73(99.97%),CD105(99.30%),CD44(99.66%),CD45 / CD34 / CD11 b / CD19 / HLA-DR(0.55%);D-The surface antigens of PDLSCs are: CD90(99.92%),CD73(99.98%),CD105(99.33%),CD44(99.70%),CD45 / CD34 / CD11 b / CD19 / HLA-DR(0.34%);P-The surface antigens of PDLSCs are: CD90(99.33%),CD73(99.56%),CD105(98.52%),CD44(99.18%),CD45 / CD34 / CD11 b / CD19 / HLA-DR(0.41%);DP-PDLSCs The surface antigens were: CD90(99.83%),CD73(99.97%),CD105(99.29%),CD44(99.57%),CD45/ CD34 / CD11 b / CD19 / HLA-DR(0.04%).The experimental samples were taken from1/3 of the periodontal ligament tissue in the roots of the patient’s teeth.Based on the results of the material location and phenotypic molecular identification,it was confirmed that the cells cultured in our experiments were PDLSCs.3.Detection of the proliferation capacity of periodontal ligament stem cells: The clone formation rates of H-PDLSCs,D-PDLSCs,P-PDLSCs,and DP-PDLSCs were 39.3 ±1.8%,31.7 ± 3.1%,23.5 ± 1.2%,16.8 ± 2.5%.respectively.Compared with the H-PDLSCs group,the clone formation rate of D-PDLSCs,P-PDLSCs,and DP-PDLSCs were all reduced,and the difference was statistically significant(P <0.05).The clone formation rate of the DP-PDLSCs group was higher than that of D-PDLSCs(P<0.05)and P-PDLSCs(P<0.05)were both low,and the difference was statistically significant.The CCK-8 method was used to test the proliferation of periodontal ligament stem cells for seven consecutive days.The results showed that the proliferation of DP-PDLSCs was lower than that of D-PDLSCs,P-PDLSCs,and H-PDLSCs,and the differences were statistically significant(P<0.05).;After 4 groups of cells(H-PDLSCs,D-PDLSCs,P-PDLSCs,DP-PDLSCs)were added with 3-MA or Rapa for drug intervention,the proliferation ability of PDLSCs in each group did not change significantly,and the differences were not statistically significant.Significance(P> 0.05).4.Transmission electron microscope results show: Compared with the H-PDLSCs group,the number of autophagosomes in the D-PDLSCs group,the P-PDLSCs group,and the DP-PDLSCs group was increased,and the destruction of intracellular organelles was more serious.The DP-PDLSCs group had the largest number of autophagosomes;No autophagosomes were found in the H-PDLSCs and H-PDLSCs + 3-MA group,and a small number of autophagosomes were found in the H-PDLSCs + Rapa group;Compared with the D-PDLSCs group,the number of autophagosomes in the D-PDLSCs + 3-MA group was reduced,and the number of autophagosomes in the D-PDLSCs + Rapa group was not significantly increased,but more autophagolysosome were found in the cells;Compared with the P-PDLSCs group,the number of autophagosomes in the P-PDLSCs + 3-MA group was reduced,and the number of autophagosomes in the P-PDLSCs + Rapa group was increased;Compared with the DP-PDLSCs group,the number of autophagosomes in the DP-PDLSCs + 3-MA group decreased,and the number of autophagosomes in the DP-PDLSCs + Rapa group increased.5.RT-q PCR results show: Compared with the H-PDLSCs group,the expression levels of the autophagy-related genes Beclin-1 and LC3 Ⅱ in the D-PDLSCs group,P-PDLSCs group,and DP-PDLSCs group increased sequentially(P<0.05),and the expression levels of P62 decreased in order(P<0.05);Compared with the H-PDLSCs group,the expression levels of autophagy genes Beclin-1 and LC3 Ⅱ in the H-PDLSCs + 3-MA group were reduced(P<0.05),the expression level of P62 increased,but there was no significant difference(P>0.05),the level of autophagy gene Beclin-1 in the H-PDLSCs + Rapa group increased,but there was no statistical significance(P>0.05),the expression level of LC3Ⅱincreased(P<0.05),the expression level of P62 decreased(P<0.05);Compared with D-PDLSCs group,the expression of autophagy genes Beclin-1 and LC3Ⅱ in D-PDLSCs+ 3-MA group were reduced(P<0.05),and the expression level of P62 was increased(P<0.05),in the D-PDLSCs + Rapa group,the expressions of Beclin-1 and LC3 Ⅱincreased(P<0.05),while the expression of P62 decreased(P<0.05);Compared with the P-PDLSCs group,Beclin-1 and LC3Ⅱ expressions were decreased in the P-PDLSCs +3-MA group(P<0.05),while the expression of P62 was increased(P<0.05),the expression of Beclin-1 and LC3 Ⅱ increased in the P-PDLSCs + Rapa group(P<0.05),while the expression of P62 decreased(P<0.05);Compared with the DP-PDLSCs group,the expression of autophagy genes Beclin-1 and LC3Ⅱ in the DP-PDLSCs + 3-MA group were reduced(P<0.05),and the expression level of P62 was increased(P<0.05),the expression of Beclin-1 and LC3 Ⅱ in DP-PDLSCs + Rapa group increased(P<0.05), while the expression of P62 showed the opposite trend(P<0.05).Conclusion:1.Compared with PDLSCs from healthy sources,PDLSCs from diabetes and chronic periodontitis showed no significant difference in morphology.All PDLSCs from diabetes and chronic periodontitis presented long-spindle fibroblast like morphology,all of which had the characteristics of stem cells,but their proliferation ability was weakened.2.3-MA and Rapa have no significant effect on the proliferation of H-PDLSCs,D-PDLSCs,P-PDLSCs,and DP-PDLSCs;3.PDLSCs in patients with type 2 diabetes with periodontitis may have excessive autophagy and organelle destruction,and the autophagy disorders in PDLSCs in diabetes and inflammatory environments may be related to severe periodontal damage in patients with type 2 diabetes. |
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