| The change of epigenetics plays an important role in the regulation of gene expression in organisms,and the process of aging in organisms is accompanied by epigenetic changes such as DNA methylation and demethylation modification.Senescence and methylation modification both are interacted.Although mesenchymal stem cells?MSCs?have a strong ability to proliferate in vitro,after a limited number of passages,MSCs show a certain diffdifferences of senescence characteristics,and their morphological,biological characteristics and epigenetic features.Both the senescence of cells and the epigenetic changes of the proliferation process add to difficulties and uncertainties in the clinical application of MSCs.Therefore,we hope to observe the changes of DNA methylation during the process of senescence of mesenchymal stem cells and the changes of DNA methylation during in vitro proliferation.The effects of the changes provide the basis for epigenetics for the"immortalization"of mesenchymal stem cells.This project will isolate and extract mesenchymal stem cells from human bone marrow for culture and passage.The senescence characteristics was observed during the passage,and a method for the determination of 5-methyl deoxycytidine?5mdC?and 5-hydroxydeoxycytidine?5hmdC?was established through the establishment of ultra performance liquid chromatography-tandem mass spectrometry?UPLC-MS/MS?.The changes of the whole genome DNA methylation and demethylation of mesenchymal stem cells during in vitro proliferation provide theoretical basis for the epigenetic analysis of mesenchymal stem cells senescence.Part? Isolation of mesenchymal stem cells from human bone marrow for subculture and evaluation of biological characteristicsObjective:To establish an efficient,simple and easy-to-use method for the isolation and culture of MSCs and to evaluate their biological characteristics through exploratory studies,and to provide a methodological and experimental basis for the standardized preparation of MSCs in clinical applications.Methods:Combining density gradient centrifugation with cell adherence and optimizing its procedures to achieve the goal of standardizing the isolation of MSCs from bone marrow and in vitro culture.The surface antigens CD29,CD34,CD44,CD45,CD73,CD90,CD105,and CD117 were detected by flow cytometry,and the cells were induced to differentiate into adipogenic,osteogenic,and chondrogenic cells to evaluate the method of isolation and culture to evaluate the quality of the mesenchymal stem cells.Result:?1?Mononuclear cells were successfully isolated using Ficoll separation solution by cell density gradient centrifugation,and human bone marrow mesenchymal stem cells were purified from the human mesenchymal stem cell whole medium in combination with cell adhesion method and successfully cultured in vitro.?2?P3 cells were successfully induced to differentiate by adipogenic,osteogenic,and chondrogenic differentiation media,and differentiation was confirmed by specific staining.?3?The positive rate of surface marker CD29 in MSCs was?99.41%±0.15%?,the positive rate of CD44?93.82%±3.37%?,the positive rate of CD73?98.32%±1.25%?,and the positive rate of CD90?95.22?.%±0.35%),CD105 positive rate?91.78%±1.80%?,while only a small amount of CD34,CD45,CD117 expression,respectively?0.23%±0.06%?,?0.13%±0.04%?and?0.25%±0.05%?.Conclusion:Human bone marrow mesenchymal stem cells can be obtained by performing density gradient centrifugation on human bone marrow to extract mononuclear cells after fibrin-cultured culture with special MSCs medium.The mesenchymal stem cells extracted by this method meet the identification criteria proposed by the International Cell Therapy Association.Part ? A method for the determination of 5-methyl deoxycytidine?5mdC?and 5-hydroxydeoxycytidine?5hmdC? in whole genome DNA by ultra performance liquid chromatography-tandem mass spectrometry ?UPLC-MS/MS?Objective:To determine the 5-methyl deoxycytidine?5mdC?and5-hydroxydeoxycytidine?5hmdC?levels of genomic DNA by ultra-high performance liquid chromatography tandem mass spectrometry?UPLC-MS/MS?.Based on the improved sample pretreatment method,the methodology is more maneuverable and provides a technical basis for observing the state of methylation and demethylation of bone marrow mesenchymal stem cells.Methods:The genomic DNA in MSCs was extracted by a spin column method and subjected to enzymatic hydrolysis to degrade the DNA into single deoxynucleosides.A method for the detection of 5-methyl deoxycytidine?5mdC?,5-hydroxymethyldeoxycytidine?5hmdC?and 2'-deoxyguanosine?dG?by UPLC-MS/MS was established.By using standard DNA to configure standard calibration curves and quality control samples,a minimum quantification limit,linear range,intraday,inter-day precision,and accuracy and stability were investigated for UPLC-MS/MS methodologies.Result:?1?5?g to 10?g of DNA can be obtained by using the QlAamp DNA Mini Kit QIAGEN kit to extract DNA from a 25 mL culture flask with bone marrow mesenchymal stem cell fusion rate of 80%to 90%.?2?Degradase buffer and DNA Degradase were used to digest 1?g DNA sample for 1 hour to completely degrade the DNA.?3?UPLC-MS/MS method for detecting 5-methyl deoxycytidine?5mdC?,5-hydroxymethyldeoxycytidine?5hmdC?and 2'-deoxyguanosine?dG?was validated.The methylation rate and hydroxymethylation rate of 5.00×10-4 can be detected in 1?g DNA.The coefficient of variation of intraday and interday precision was?10%.Accuracy is between 98%and 104%.The linear correlation coefficient is greater than0.995.Placement at room temperature for 2 hours,autosampler for 4 hours,refrigerator at 4°C for 7 days and-20°C for 30 days did not affect detection.Conclusion:TheUPLC-MS/MSgenomicDNAmethylationand hydroxymethylation detection methods established in this study have good operability,accuracy and precision and can be observed.The state of methylation and demethylation during the passage of bone marrow mesenchymal stem cells provides the technical basis.Part ? Senescence changes of mesenchymal stem cells in vitro and changes of DNA methylation and hydroxymethylation in whole genomesObjective:Observe changes in DNA methylation and hydroxymethylation of mesenchymal stem cells subcultured in vitro,and monitor their aging process and performance to understand DNA methylation and hydroxymethylation of mesenchymal stem cells during senescence.Methods:Ten cases of human bone marrow mesenchymal stem cells were extracted and isolated,and they were continuously passaged until the proliferation ability of the cells disappeared.P1,P3,P5,P7,P9 and P13 cells were stained by senescence-associated?-galactosidase staining.The RNA and DNA of the P1,P5 and final passage cells were collected to compare the Oct4 mRNA expression level and the whole genome DNA methylation and methylolation levels.Whole-genome DNA methylation sequencing was performed on three of the first and last passage cells.Result:?1?The senescence-associated?-galactosidase staining was performed on the P1,P3,P5,P7,P9 and P13 bone marrow mesenchymal stem cells.The results showed that the positive staining rates were 2%,11%,35%,and 72%,respectively.,86%and 94%.The positive rate of?-galactosidase staining increased from passage to passage.?2?The relative expression levels of Oct4 mRNA in the P1,P5 and last generation cells decreased with passage of cells.The expression level of Oct4 mRNA in P1 was higher than that in the fifth passage,p<0.05;and the expression of Oct4mRNA was significantly decreased in the last passage compared with the fifth passage,p<0.01.?3?10 cases of bone marrow mesenchymal stem cells detected methylation and hydroxymethylation results showed that the average of the genome-wide methylation rates of the first,fifth,and last passage cells were 2.52%,1.81%,and1.61%,respectively.The methylation rate was significantly lower in the last passage cells,p<0.05.The whole genome hydroxymethylation rate did not change significantly.?4?3 cases of bone marrow mesenchymal stem cell whole-genome methylation sequencing showed that the average methylation rate of all C-sites in the first passage of cells was 3.34%,and the average methylation rate of CG-type methylation was 62.13%.The average methylation rate of CGH was 0.4%,the methylation rate of CHH type was 0.37%,and the average methylation rate of all C-sites of the last-passage cell was 2.74%.The average methylation rate of CG type was 50.62%,the average methylation rate of CHG type is 0.42%,and the average methylation rate of CHH type is 0.39%.The methylation rate of the C-site and the methylation rate of the CG-type were significantly lower in the final passage than in the first passage cells?p<0.05?.Conclusion:BMMSCs will continue to senescence during in vitro culture,and their cell differentiation ability will continue to decrease.With the increase of the number of passages,the genomic DNA hydroxymethylation levels of BMMSCs did not change significantly,while the methylation level decreased overall,among which the methylation rate of CG type was mainly reduced.This study for the first time observed the epigenetic changes in the process of subculture of BMMSCs from the perspective of whole-genome DNA methylation level,and provided the experimental basis of epigenetics for the senescence of mesenchymal stem cells.In addition,the methodological results established in this study are consistent with the sequencing results,suggesting that the detection method is practical and reliable. |
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