Transcriptomic And Proteomic Studies Of Periodontal Ligament Stem Cells In Type 2 Diabetes Mellitus With Periodontitis

Objective: The research found that the biological properties of periodontal membrane stem cells with periodontitis in type 2 diabetes mellitus changed,but the reasons for the change are not clear at present,and whether the gene and protein levels of periodontal stem cells in different inflammatory environments and the mechanism of their changes need to be further clarified.To this end,this experiment will be a single disease(type 2 diabetes or periodontitis)and periodontitis with type 2 diabetes patients for a joint study of transcriptomics and proteomics,in order to find the periodontitis,with or without type 2 diabetes signature genes and proteins,to provide a preliminary experimental basis for the early diagnosis,monitoring and prognosis of the disease.Methods: In this experiment,four groups of periodontal membrane tissues of different sources of teeth were obtained in vitro,and periodontal stem cells were obtained by enzymatic tissue block method;the experimental groupings were: healthy group(Hh PDLSCs),simple type 2 diabetes group(D-h PDLSCs),simple periodontitis group(Ph PDLSCs),and type 2 diabetes with periodontitis(DP-h PDLSCs);(1)Cells were cloned and cultured by finite dilution method to PDLSCs,flow cytometry to detect relevant phenotype of cells,plate cloning and CCK-8 to detect cell proliferation;(2)The total RNA and total proteins of the four groups of samples were collected,and the RNA-Seq and LC-MS/MS methods were used to screen the differentially expressed genes and proteins in the relative healthy group of the disease group,and the GO function annotation and signaling pathway enrichment analysis of genes and proteins were carried out;(3)Data integration analysis of transcriptome and proteome,to find out the genes and proteins that are commonly up-regulated,and to analyze the protein interaction network;(4)Finally,q RT-PCR and WB were used to verify the expression of differential genes and differential proteins.Results:(1)Four groups of h PDLSCs of different origins were successfully cultivated,which were manifested as long fusiform or irregular forms.The identification of flow cytometry showed that the mesenchymal stem cell phenotypes of the four groups of h PDLSCs were all highly expressed;the results of the plate cloning experiments were as follows: H-hh PDLSCs,Dh PDLSCs,P-hp PDLSCs,and DP-h PDLSCs,the clonal formation rates respectively were34.67±1.36,27.2±1.16,24.1±1.17,11.29±1.01(P<0.05),and the CCK-8 method showed that the four groups of cells had the ability to proliferate,of which CCK-8 showed that all four groups of cells had proliferative ability The proliferation capacity of DP-PDLSCs was significantly weakened(P<0.05);(2)Transcriptome analysis: 123 upregulated genes and 113 downregulated genes compared with healthy group in the simple type 2 diabetes group,65 upregulated genes and 119 downregulated genes compared with healthy group in the periodontitis group alone,173 upregulated genes and 389 downregulated genes in the type 2 diabetes group with periodontitis compared with healthy group(threshold p-Value<0.05,|log2foldchange|>0.0);there are 31 different genes in the three groups,and there are 402 different genes specific to type 2 diabetes mellitus with periodontitis,of which the characteristic difference genes in the type 2 diabetes with periodontitis group are: TLR2,NOD2,IL12RB2,CD14,ADM,APOE,THSD4,and significantly enriched in apoptosis,vascular endothelial growth factor signaling pathway,pantothenic acid and Co A biosynthesis.Gn RH signaling pathway,Erb B signaling pathway,oxidative phosphorylation,autophagy and other pathways.(3)Proteome analysis: 149 up-regulated proteins and 33 down-regulated proteins were compared with the healthy group in the simple type 2 diabetes group;221 up-regulated proteins and 47 down-regulated proteins were compared with the healthy group;389 upregulated proteins and 265 down-regulated proteins were compared with the healthy group in the type 2 diabetes group with periodontitis(threshold p-Value <0.05,|log2foldchange|>1.5),among which the characteristic differential proteins in the group of type 2 diabetes mellitus with periodontitis were: VPS4 A,CASP3,TIMP1,CD47,RAB5 C,and were significantly enriched in pantothenic acid and Co A biosynthesis,tumor necrosis factor signaling pathway,Toll-like receptor signaling pathway,type 2 diabetes mellitus,NF-κB signaling pathway and other pathways.(4)Through the combined analysis of transcriptome and proteome,the genes of differential genes and differential protein trends were screened out,and the common differential genes and differential proteins of periodontitis and type 2 diabetes with periodontitis were identified: CD44,ANPEP,HSPE1.(5)q RT-PCR and Western Blot showed that Rab5 C was upregulated in the periodontitis and type 2 diabetes with periodontitis,more significantly in the type 2 diabetes with periodontitis group,and q RT-PCR was confirmed by the Western Blot results,which was better consistent with the sequencing results.(P<0.05)Conclusion:(1)In the presence of type 2 diabetes or periodontitis alone or both sometimes the biological capacity of the periodontal ligament stem cells is negatively affected;(2)The comprehensive data of transcriptome and proteome show that CD44,ANPEP and HSPE1 with differential expression of type 2 diabetes mellitus with periodontitis can be used as candidate marker genes for the diagnosis and treatment of periodontitis and type 2diabetes with periodontitis;(3)Pantothenic acid and Co A biosynthesis,endocytosis signaling pathway and RAS signaling pathway were finally screened by co-enrichment pathways of differential genes and differential proteins,laying the foundation for subsequent mechanism studies,and the expression of the gene Rab5 C in type 2 diabetes with periodontitis was significantly higher than that in the control group,suggesting that there was a relationship between Rab5 C and type 2 diabetes with periodontitis.