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Observation Of Cytobiological Characteristics And Mechanism Of Adipose-derived Stem Cells In A Murine Model Of Systemic Lupus Erythematosus

Posted on:2021-06-04Degree:MasterType:Thesis
Country:ChinaCandidate:S D XieFull Text:PDF
GTID:2544306035977839Subject:Dermatology and venereology
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Systemic lupus erythematosus is a chronic autoimmune disease.Traditional treatment has a variety of side effects,which have a great impact on the quality of life of patients.Stem cell therapy is a new treatment.Adipose-derived stem cell(ADSC)is one kind of pluripotent stem cells with the characteristics of high self-renewal,multi-directional differentiation,immunomodulatory and weak immunogenicity.More and more studies have confirmed that ADSC can release and treat of autoimmune diseases.Some studies have found that SLE bone marrow mesenchymal stem cells(BMSCs)have basic attribute defects,and autologous BMSC transplantation is not effective in the treatment of SLE.At present,there is no study on whether SLE Adipose-derived stem cells can maintains normal biological and functional characteristics.Mammalian rapamycin target protein(mTOR)participates in many life activities such as cell protein synthesis,cell proliferation,growth,cell metabolism,et al.Our previous studies found that mTOR pathway is over-activated in the kidney of lupus mice,which is involved in the pathogenesis of lupus,so it is speculated that it may be related to the morbidity of mesenchymal stem cells.ObjectiveThe purpose of this study was to observe the biological characteristics of adipose-derived mesenchymal stem cells,isolated and cultured in vitro from a mouse model of systemic lupus erythematosus(MRL/lpr),including morphology,proliferation,migration,apoptosis,senescence,autophagy and AKT/mTOR signal pathway.Compared with normal C57 mouse ADSC,the cytobiological characteristics and related mechanisms were analyzed,in order to provide experimental basis for clinical application of adipose mesenchymal stem cells.Methods1.The primary generation of ADSCs were isolated from normal C57 mice and MRL/lpr mice by using type I collagenase and cultured to the passage 3 in vitro for the follow-up experiments.2.The morphology of mouse ADSCs was observed under ordinary optical microscope,and the cell growth curve was detected by CCK-8.3.Cell scratch experiment and Transwell migration assay were used to detect the migration ability of ADSC in each group.The apoptosis of ADSCs were evaluated by FCM with AnnexinⅤ FITC-PI staining.β-galactosidase assay was used to detect cell senescence.4.The proteins of LC-3,Beclin-1,p62,P21,p5 3,mTOR,p-mTOR,Akt and p-Akt in the third generation of ADSC were detected by Western blot.StatisticsThe statistical software SPSS20.0 was applied for data analysis,the results expressed as mean±standard deviation(x ± SD).Statistical method:the quantitative data between the two samples were analyzed by Student’s t-test,or analysis of variance.A value of P<0.05 was considered statistically significant difference.Results1.ADSCs of mouse were cultured and subcultured Successfully.In about 6-7 days,the stem cells reached up to 90%fusion.After trypsin digestion,theproliferation rate of ADSCs was faster than that of the primary generation,and the cells could be proliferated and passaged stably.The morphology of these cells was typical fusiform,arranged in reticulating and swirling.ADSCs from C57 mice and MRL/lpr mice aged 4-6 weeks were fusiform similarly,while the ADSCs from MRL/lpr mice aged 18-20 weeks were mostly fusiform,with cell volume increased,and numbers of refractive round cells and polygonal cells can be seen.2.The cell growth curve showed that the proliferation ability of ADSCs from 4-6-week-old C57 mice and MRL/lpr mice was strong,and the growth curve was in the shape of "S",with typical latent period,rapid proliferation period and platform phase.However,the proliferation ability of ADSCs from the older MRL/lpr mice aged 18-20 weeks was weak,only had a certain proliferation ability in the first 3-4 days,and the high peak value was lower than that of the mice aged 4-6 weeks,and then the growth rate decreased gradually.3.The results of Cell scratch experiment showed that 24 hours after scratching,the relative migrated distance of ADSCs from 4-6-week-old MRL/lpr mice was lower than that of the C57 mice(p<0.05),but higher than that of ADSCs from MRL/lpr mice aged 18-20 weeks(p<0.05).The results of Transwell migration assay were consistent with cell scratch wound healing assay.4.The apoptosis of ADSCs in the passage 3 was similar between from C57 mice and MRL/lpr mice aged 4-6-week,and there was no significant difference.The senescence staining results of ADSCs of the two groups were also similar.But the stained cells increased in the ADSCs from 18-20-week-old lupus mice.5.The expression of senescence-related protein p53 and autophagy-related proteins such as LC3-Ⅱ,Beclin-1 and p62 increased in ADSCs of the lupus group when compared with those in the C57 group aged 4-6 weeks.No significant difference in the expression of mTOR pathway-related proteins,such as mTOR,p-mTOR,Akt and p-Akt between the ASDCs of these two groups.ConclusionCompared with C57 mice,the ADSCs of MRL/lpr mice are abnormal:migration ability decreased,active autophagy,and expression of aging related protein P53 increased,but there was no significant difference in the expression of mTOR pathway,suggesting that some of the cellular characteristics and functions of ADSCs in systemic lupus erythematosus are abnormal,with a tendency of premature senility,which are not significantly related to the mTOR pathway.Compared with the ADSC of 4-6-week-old MRL/lpr mice,the ADSC from MRL/lpr mice aged 18-20 weeks showed changes in cell morphology,proliferation and migration ability decreased,and senescence increased,suggesting that the quality of ADSC may decline with the increase of animal age.
Keywords/Search Tags:Systemic lupus erythematosus, Adipose mesenchymal stem cells, migration, autophagy, senescence
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