| Objective:The interaction between diabetes and periodontitis is still attracting attention.Patients with type 2 diabetes and periodontitis have a high degree of damage to the periodontal tissue,rapid progress,and poor prognosis after treatment.Hyperglycemia in patients with type 2 diabetes often induces oxidative stress in the body,which affects the signal pathways in the body,and induces cell activity and even death in the body.Human periodontal ligament stem cells(hPDLSCs)are used as seed cells for periodontal regeneration.Whether they can maintain their stem cell activity in the environment of type 2 diabetes with periodontitis needs further clarification.This experiment intends to explore the interaction between oxidative stress,apoptosis and autophagy of periodontal ligament stem cells from patients with type 2 diabetes with periodontitis,and further explore the relationship between type 2diabetes and periodontitis.Methods:In the experiment,the periodontal ligament tissues of healthy individuals,simple type 2 diabetes patients,simple periodontitis patients,and type 2 diabetes with periodontitis were collected in vitro,and human periodontal tissues were obtained by cloning and purification using the enzymatic tissue block method and the limiting dilution method.Membrane stem cells,and use flow cytometry for molecular identification of cell phenotype.The experiment was divided into experimental groups: healthy group(H-hPDLSCs),simple diabetes group(D-hPDLSCs),simple periodontitis group(P-hPDLSCs),diabetes with periodontitis group(DP-hPDLSCs);inhibitor group(Intervention with inhibitor SP600125 to form four inhibitor groups):blocker-H-hPDLSCs,blocker-D-hPDLSCs,blocker-P-hPDLSCs,blocker-DP-hPDLSCs.1)Perform the following experiments with the experimental group:(1)CCK-8 to detect cell proliferation;(2)Plate cloning to detect cell self-renewal;(3)Osteogenesis,adipogenesis,and chondrogenesis are induced for 21 days and stained;(4)Flow cytometry to detect ROS level;(5)Double fluorescence staining to detect cell apoptosis;(6)Immunofluorescence to detect cell LC3 autophagy protein;(7)Transmission electron microscopy to detect autophagosomes and mitochondria;(8)RT-PCR to detect cell autophagy and apoptosis-related gene expression;(9)Western blot to detect cells Autophagy and apoptosis-related protein expression.2)The following experiments were carried out with the experimental group and the inhibitor group(8 groups in total):(1)Immunofluorescence detection of cell LC3 autophagy protein;(2)Double fluorescence staining to detect cell apoptosis;(3)RT-PCR detection of cell autophagy and apoptosis related Gene expression;(4)Western blot to detect cell autophagy,apoptosis and pathway-related protein expression.3)Results:1)(1)Successfully completed the in vitro culture of hPDLSCs from different sources,cloned and purified by the limiting dilution method,the four groups of cells showed no significant difference in morphology;flow cytometry was used to identify the cell phenotype and the results showed that the surface antigen CD90 of the four groups of hPDLSCs,CD44,CD105 are all highly expressed,and isotype control antigens CD45/CD11b/CD19/CD34/HLADR are all low expressed;(2)The results of the low-density seed plate cloning experiment are as follows: H-hPDLSCs,D-hPDLSCs,P-hPDLSCs,DP-hPDLSCs The clone formation rates of the four groups were: 40.3± 1.5%,33.4 ± 2.3%,30.1 ± 1.2%,17.2 ± 1.7%(P<0.05);CCK8 method was used to detect the OD value of hPDLSCs for 7 consecutive days.The results showed that the four groups of cells have the ability to proliferate.Among them,the proliferation ability of DP-PDLSCs was the lowest(P<0.05);(3)The four groups of hPDLSCs were induced for adipogenesis,osteogenesis,and chondrogenesis for 21 days.The staining results are as follows: According to H-hPDLSCs,D-hPDLSCs,P-hPDLSCs,DP-hPDLSCs,after Alizarin Red staining,the number of mineralized nodules(ie,red precipitates)in the four groups of cells decreased sequentially,and the degree of compactness decreased sequentially;after oil red O staining,the appearance time of lipid droplets in the four groups of cells increased sequentially,and the number decreased sequentially;The staining showed that the stained area and color depth of the four groups of cells decreased in turn;(4)DCFH-DA detected the ROS level in hPDLSCs and the results showed that: compared with H-hPDLSCs,the ROS levels generated by P-hPDLSCs did not change significantly(P>0.05),D-hPDLSCs The ROS production of DP-hPDLSCs and DP-hPDLSCs both increased significantly(P ﹤ 0.01),and the ROS level in the DP-hPDLSCs group was the highest(P﹤0.01);(5)The third generation cells from four groups of different sources were immunized after double staining with Calcein AM and PI It can be seen under a fluorescence microscope that H-hPDLSCs have the least number of apoptosis,followed by D-hPDLSCs and P-hPDLSCs,and DP-hPDLSCs has the largest number of apoptosis;(6)The expression of cell LC3 protein is detected by immunofluorescence,according to H-hPDLSCs,D-hPDLSCs,The fluorescence intensity and area of P-hPDLSCs and DP-hPDLSCs increased in sequence;transmission electron microscopy detection of autophagosome and mitochondrial structure of four groups of hPDLSCs showed that: compared with H-hPDLSCs group,D-hPDLSCs group,P-hPDLSCs group,DP-The number of autophagosomes formed in the hPDLSCs group increased,among which the DP-hPDLSCs group had the largest number of autophagosomes;the H-hPDLSCs group showed most of the mitochondrial membrane and cristae intact;D-hPDLSCs,P-hPDLSCs,DP-hPDLSCs groups It can be seen that part of the mitochondrial cristae is destroyed and disappeared,showing The result of RT-PCR showed that the expression of Bcl2 and p62 decreased in the order of H-hPDLSCs,D-hPDLSCs,P-hPDLSCs,and DP-hPDLSCs.The expressions of Caspase3,LC3,and Beclin1 decreased in the order of H-hPDLSCs,D-hPDLSCs,P-hPDLSCs,DP-hPDLSCs increased in sequence,and the difference between groups was statistically significant(P<0.05);(8)Western blot results showed mutual confirmation with RT-PCR results.2)(1)After Calcein AM and PI double staining,the apoptosis of H-hPDLSCs,P-hPDLSCs and D-hPDLSCs can be seen under the immunofluorescence microscope compared with the cells after intervention(blocker-H-hPDLSCs,blocker-D-hPDLSCs,blocker-P-hPDLSCs)apoptotic number increased,and the cells in the DP-hPDLSCs group decreased compared with the cells after the intervention(blocker-DP-hPDLSCs);(2)The results of immunofluorescence detection of autophagy marker protein LC3 showed that the autophagy LC3 fluorescence of each group after the intervention The intensity is weakened.(3)Western blot results showed that compared with the experimental group,the ratio of LC3Ⅱ/LC3Ⅰ in the inhibitor group after SP600125 intervention decreased(P < 0.05),while the expression of Beclin1,Bcl2,p-Bcl2,and p-JNK decreased(P<0.05);Autophagy receptor protein p62 expression increased(P<0.05);compared with H-hPDLSCs,D-hPDLSCs,P-hPDLSCs,blocker-H-hPDLSCs,blocker-D-hPDLSCs,blocker-P-hPDLSCs apoptosis The expression of gene caspase3 increased(P<0.05),and the expression of caspase3 in the blocker-DP-hPDLSCs group was lower than that of DP-hPDLSCs(P<0.05).(4)The RT-PCR result is corroborated with it.3)Conclusion:1.Inflammation and type 2 diabetes inhibit the proliferation and multidirectional differentiation of hPDLSCs,which may have a negative impact on the biological properties of hPDLSCs.2.The high blood sugar environment stimulates hPDLSCs to generate a large amount of ROS,which may induce oxidative damage to cells;3.Both autophagy and apoptosis of hPDLSCs in patients with type 2 diabetes mellitus with periodontitis increased.4.Under the combined action of hyperglycemia and inflammation,JNK is activated by phosphorylation and induces apoptosis of hPDLSCs,which may be related to the autophagy disorder of hPDLSCs under oxidative stress. |
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