| Diabetes mellitus(T2DM)is the second major chronic disease after cardiovascular disease,with Type 2 diabetes mellitus(T2DM)accounting for 90%-95%.Diabetic hyperglycemia toxicity,abnormal insulin and cytokine levels,and oxidative stress lead to the formation of diabetic osteoporosis characterized by low bone mass and bone microstructure destruction.For oral implantation,patients with poor blood glucose control showed decreased early bone integration,prolonged healing time of surrounding soft tissue,and increased risk of infection.In recent years,the discovery of stem cells has brought new hope for a variety of tissue repair.Stem cells have the potential of multi-directional differentiation,self-renewal and the ability to secrete a variety of cytokines,which can participate in damaged tissue and organ repair and regeneration.Stem cells can be divided into totipotential stem cell,multipotential stem cells and unipotent stem cells.The use of stem cells in tissue engineering to improve osteogenesis in diabetic patients has also been demonstrated in several experiments.Stem cells derived from autologous cells,which are clinically available and can promote longterm survival and tolerance of transplantation,are ideal seed cells.Bone marrow mesenchymal stem cells(BMSCs)and adipose-derived stem cells(ASCs)were the most widely used.However,it is difficult to obtain BMSCs in clinical practice,and BMSCs from T2DM patients are less in number,less able to regenerate,less able to proliferate and survive,and significantly less able to differentiate into osteoblasts compared with normal BMSCs,which limits their application in the direction of regeneration.However,the number of ASCsderived from T2DM(ASCs-T2DM)did not decrease,and the growth curve was similar to that of the control group.Therefore,ASCs has become a more promising cell.However,compared with ASC derived from control rats(ASCs-Control,ASCs-C),the osteogenic ability of ASCs-T2 DM is weaker,which cannot meet the needs when applied to bone tissue repair.The reason is that the body environment of T2 DM can change the osteogenesis and proliferation of ASCs.In other words,for patients with T2 DM,the different state of the body before and after the disease will also affect the gene expression,but the individual's genetic sequence has not changed,and this phenomenon may be related to epigenetics.DNA methylation is the most widely studied part of epigenetics.DNA methylation in gene promoter region often inhibits gene expression.Considering the instability before and after modeling when studying individuals,we chose to study the differences in DNA methylation levels between ASCs-T2 DM and ASCs-C in gene promoter region,looking for differential genes,so as to clarify the specific mechanism of the influence of T2 DM body environment on the osteogenic ability of ASCs.Part ?: Comparative study of ASCs-T2 DM and ASCs-CObjective: To establish a rat model of T2 DM and compare the osteogenesis and proliferation of ASCs-T2 DM and ASCs-C in vitro.Methods: The T2 DM model was established by high-fat and high-sugar diet combined with low-dose STZ intraperitoneal injection.After successful modeling,ASCs were extracted from healthy and type 2 diabetic rats by enzyme digestion,and cultured into P3 cells for later use.Flow cytometry was used to detect the expression of cell surface molecules.Clonal formation ability was detect by clonal formation assay;CCK-8 method was used to detect cell proliferation.The induction of osteogenic lipids verified the ability of multidirectional differentiation.The expression of osteogenic related genes was detected by q-PCR,and the expression of osteogenic related proteins was detected by Western Blot.Results: After modeling,the body weight of rats decreased,and the blood glucose was continuously maintained higher than 16.7mmol/L.Flow assay showed that both cells positively expressed stem cell related surface markers(CD29 and CD90),but not blood cell related surface markers(CD34 and CD45).Compared with ASCs-C,the clonal formation ability and proliferation ability of ASCs-T2 DM were significantly decreased.After lipogenic induction,staining showed that both stem cells could produce lipid droplets.The expression of ALP in ASCs-T2 DM was significantly lower than that of ASCs-C.Alizarin red staining showed that fewer mineralized nodules were formed in ASCs-T2 DM.q-PCR and Western Blot results showed that the expression of osteogenic related genes and proteins in ASCs-T2 DM was also lower than that of ASCsC.Conclusion: The proliferation,clonal formation and osteogenic differentiation of ASCsT2 DM were significantly lower than that of ASCs-C.Part ?: DNA methylation sequencing of ASCs-T2 DM and ASCs-C whole genome and screening of differentially expressed genesObjective: To screen the promoter DNA methylation differentially expressed genes related to osteogenesis in ASCs-T2 DM and ASCs-C.Methods: DNA of ASCs-T2 DM and ASCs-C were extracted to DNA methylation sequencing.GO term,which was related to osteogenesis and whose p value was less than 0.05,was selected to select the related genes.Combined with DNA methylation and q-PCR analysis,genes with negative correlation between methylation level and gene expression level were screened.Combined with IGV software and literature reading,the target gene was determined.Results: Genome-wide methylation results showed that DNA methylation levels did differ between the two groups.There were 253 differentially methylated genes in the promoter region.The GO term genes associated with osteogenesis with p value less than 0.05 were C3(complement C3),Calca(calcitonin-related polypeptide),Ereg(epiregulin)and il20rb(interleukin 20 receptor subunit beta).The expression levels of C3 and Calca were negatively correlated with DNA methylation.Finally,the gene with the most significant difference was identified: Calca.Conclusion: There is a difference between ASCs-T2 DM and ASCs-C in DNA methylation level of promoter region.Among them,the DNA methylation level of Calca in ASCs-T2 DM was higher than that of ASCs-C,while its expression level was lower than that of ASCs-C.Part ?: Verifying the function of Calca in promoting osteogenic differentiation of ASCs-T2DMObjective: To investigate the effect of Calca gene coding product CGRP on the morphology,proliferation and osteogenesis of ASCs-T2 DM.Methods: Complete medium and osteogenic induction medium containing different concentrations of CGRP(10-7mol/L,10-8 mol/L,10-9 mol/L,0 mol/L)were prepared.The influence of CGRP on cell morphology was detected by phallopeptide staining.CCK-8 was used to detect the effect of CGRP on cell proliferation.After osteogenic induction staining,the effects of CGRP at different concentrations on the osteogenic capacity of ASCs-T2 DM in vitro were detected.The effect of ASCs-T2 DM on the expression of osteogenic genes was detected by q-PCR.Results: CGRP had no effect on cell morphology.However,it can affect the proliferation of ASCs in T2 DM rats.10-7-10-8mmol/L concentration medium can significantly improve the proliferation of ASCs.ALP staining showed that: with the intervention of this factor,the expression of ALP was significantly increased,and it has a concentration dependent tendency.Alizarin red staining showed that CGRP could promote the formation of calcium nodules of ASCs-T2 DM,which was also concentration-dependent.q-PCR results showed that the expression of osteogenic related genes increased significantly under the intervention of CGRP.Conclusion: To a certain extent,CGRP does not affect the morphology of ASCsT2 DM,but can improve its osteogenic differentiation and proliferation in vitro.Part ?: The locus and functional verification of Calca gene DNA methylation changesObjective: To reversely verify the correlation between the methylation level of Calca gene promoter DNA target fragment and gene expression in ASCs-T2 DM,and the specific mechanism was studied.Methods: 5-Azacytidine was used to interfere with ASCs-T2 DM in vitro,and the effect of 5-Azacytidine on cell morphology was detected by Phalloidin staining.Bisulfite amplicon sequencing was used to detect the effects of 5-Azacytidine on DNA methylation of ASCs-T2 DM target fragments.The expression of Calca and DNMT1 in ASCs-T2 DM was detected by q-PCR after the intervention of 5-Azacytidine.Results: 5-Azacytidine could significantly change the morphology of ASCs-T2 DM.Bisulfite amplicon sequencin results showed that 5-Azacytidine could reduce the methylation level of Calca gene DNA promoter in ASCs-T2 DM.q-PCR showed that the expression of Calca in ASCsT2 DM was increased after 5-Azacytidine interference,while the expression of methyltransferase DNMT1 was decreased.Conclusion: 5-Azacytidine can reduce the DNA methylation level of the target fragment in the promoter region of Calca gene in ASCsT2 DM.This process may be mediated by reducing the expression of DNMT1,thereby improving the expression of Calca gene. |
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