| Objective:On the basis of systematic screening,evaluation and acquisition of an elderly macaque model,the elderly multiple organ dysfunction model was used to investigate the effects of juvenile bone marrow mesenchymal stem cells(BMSC)on the thymus tissue structure and expression of related factors influences.Through transcriptome sequencing of thymus tissues,we explored the mechanism of BMSC on thymus atrophy in senile macaques,and explored the feasibility of BMSC to delay or reverse thymus atrophy,and provide a theoretical basis for clinical BMSC treatment of thymus degeneration related to immune system aging.Methods:1.Screening of aging macaque models:5 healthy young female macaque monkeys,with an average age of 3 years old and weighing 2～3kg;10 elderly female macaque monkeys,with an average age of 25 years old and weighing 4～5kg.2.Isolation,cultivation and identification of BMSC:Bone marrow puncture was used to collect bone marrow of healthy young female macaque monkeys with an average age of 3 years under sterile conditions.BMSC were isolated and purified by differential adherence and subculture,and were passaged 4 times amplify to obtain BMSC.Observe the morphology and growth status of BMSCs under an inverted phase contrast microscope.Flow cytometry detected the positive expression ratio of CD29,CD34,CD90 and CD105 cell surface antigens,and induced BMSC to differentiate into adipogenic,osteogenic and chondrogenic cells in vitro Differentiation ability.3.Animal grouping and BMSC transplantation treatment:The above screening models were randomly grouped,namely 5 in the juvenile group(n=5),4 in the elderly group(n=4)and 6 in the elderly treatment group(n=6).Macaque monkeys in the elderly treatment group were transplanted with BMSCs through the femoral vein at a dose of1×10~7cells/kg,and were infused once every other day for 3 consecutive times;macaque monkeys in the juvenile group and the elderly group were infused with the same volume of saline at the same time.Routine rearing,each group of macaque monkeys was sacrificed 6 months after the last transplantation,and materials were collected.4.Evaluation method of the therapeutic effect of BMSC transplantation treatment:PET-CT was used to observe the changes of thymus imaging before treatment,3months and 6 months of treatment in the young group and the elderly treatment group;after 6 months of BMSC treatment,anesthesia the macaque monkeys of each group were sacrificed,thymus tissues were collected,observed and photographed;the thymus glands of each group were weighed to calculate the thymus index;a part of the thymus tissues were collected,fixed with 4%paraformaldehyde solution,paraffin-embedded sections,and stained with HE,Masson and Immunofluorescence staining to observe changes in thymus tissue structure,collagen fiber deposition,and changes in thymus cortex and medulla structure;flow cytometry was used to detect T-cell subsets and regulatory T cells(Tregs)in the peripheral blood of macaque monkeys changes in cell output levels;ELISA method was used to analyze the secretion levels of monkey thymosinαand monkey thymosin II in the peripheral blood of macaque monkeys in the elderly treatment group.5.Evaluation method of the mechanism of BMSC transplantation treatment:analysis of the expression of senescence-related proteins(P21,P53,Sirt1 and Sirt3)and apoptosis-related proteins(Caspase3,Bax and Bcl-2)by immunohistochemical staining;observation of each by Tunel staining Apoptosis of thymus cells in macaque monkeys;next-generation sequencing technology was used to sequence m RNA in thymus tissues of macaque monkeys,and sequencing data was analyzed using bioinformatics methods to compare the differences in transcripts of macaque monkey m RNAs between different ages and screen for aging-related Genes;through 3D PCA,cluster analysis and trajectory analysis,explore the changes of aging-related genes before and after treatment;use bioinformatics methods to screen out aging-related genes that show differential changes after treatment,and analyze their main through GO enrichment analysis Enriched pathway.Results:1.Through evaluation and screening of aging macaque monkeys,successfully obtained aging phenotypes.The elderly cynomolgus monkeys have aging phenotypes,bleak hair color,poor mobility,and have typical characteristics of brain atrophy,pulmonary fibrosis,thymus and ovarian atrophy.2.The BMSCs were isolated and cultured by the differential adherence method.BMSCs began to grow in 3 to 4 days,and the primary BMSC cells grew in colonies.When the cell fusion rate reached 80%to 90%,the cells were expanded and passaged.The fourth-generation(P4)cells had a uniform morphology,long fusiform,fibroblast-like,and vortex-like growth.After differentiation ability test,oil red O staining was positive after P4 generation BMSCs induced adipogenic differentiation;by flow cytometry,the positive rates of CD29,CD34,CD90 and CD105 were found to be 98%,0.99%,98.8%,99.8%,which meets the standard of mesenchymal stem cells;after osteogenic induction and differentiation culture,alizarin red staining is positive;after cartilage induction and differentiation culture,alison blue staining is positive.3.Evaluation of the efficacy of BMSC transplantation treatment:Observed by PET-CT,PET-CT of the thymus in the young group and the elderly group showed normal thymus position.Most of the thymus was replaced by fat tissue before treatment in the elderly group,and only a small amount of fine fiber cord strip structure was seen.Density is the same as adipose tissue,SUVmax<1;3 months and 6 months after treatment,the thymus tissue density gradually increases,CT value gradually increases,SUVmax>1;thymus index,the elderly group is significantly lower than that of the young(P<0.01)Rhesus monkeys in the group were higher than the elderly group(P<0.05),but there was no significant difference between the elderly treatment group and the younger group(P>0.05);HE staining showed that the thymus was intact in the juvenile group.The thymus gland of rhesus macaques in the elderly group was atrophic,and the parenchymal area of the thymus gland was reduced,and the atrophic area was up to 90%.Rhesus macaques in the treatment group showed continuous changes to normal thymus tissue,and the area of thymus gland reached more than 30%;through HE staining,it was found that the thymus tissue structure and capsule were relatively complete in the young group,and the elderly treatment group was older The thymus tissue structure of the group was complete,the thymus parenchyma area increased,the skin and medulla structure improved and began to appear boundaries,the thymus cells increased,and the adipose tissue decreased;by Masson staining,it was found that the degree of thymus fibers in the young group was low,and the elderly treatment group had collagen Fibre improvement,collagen hyperplasia is lower than that of the elderly group,and the degree of thymus fibrosis is significantly reduced;through immunofluorescence staining,it was found that the junction structure of cortex and medulla gradually appeared in the elderly treatment group,and the thymus structure was dense.The CK5 and CK8 elderly treatment groups expressed more expression Significantly increased(P<0.05);CK5+CK8 elderly treatment group CK5 expression was significantly higher than the elderly group(P<0.05),CK8 expression has an upward trend than the elderly group(P>0.05);Group testing found that the percentage of CD3~+T cells in the peripheral blood showed a trend of increasing first and then decreasing.The 30-day and 60-day treatments were significantly higher than before treatment(P<0.05),and the 90-day treatment was lower than before treatment(P>0.05);CD3~+CD4~+T cells showed a trend of slow growth,there was no statistically significant difference before treatment compared with 30 days,60 days and 90 days of treatment(P>0.05);CD3~+CD8~+T cells showed a trend of increasing first,then decreasing and then increasing,treatment 30 days,After 60 days and 90 days of treatment,there was no statistical significance compared with before treatment(P>0.05);through the detection of Tregs in peripheral blood of cynomolgus monkeys in the elderly treatment group,the percentage of Tregs in peripheral blood showed a trend of first decreasing and then increasing after treatment.Among them,30 days of treatment,60 days of treatment and 90 days of treatment were significantly more significant than before treatment(P<0.05);the thymosinαand thymopoietin II secretion in peripheral blood of cynomolgus monkeys in the elderly treatment group was analyzed by ELISA It was found that the level of thymosinαsecretion increased first and then decreased after BMSC infusion.Among them,30 days of treatment increased significantly than before treatment(P<0.01),and 60 days and 90 days of treatment increased compared with before treatment(P>0.05);The secretion level of monkey thymosinⅡincreased first and then decreased.It increased after 30 days and60 days after treatment,and decreased after 90 days of treatment(P>0.05).4.Evaluation of the treatment mechanism of BMSC transplantation:by immunohistochemical staining,it was found that the expression of aging-related protein P21 in the elderly treatment group was significantly lower than that in the elderly group(P<0.05),and the expression of P53 in the elderly treatment group was significantly lower than that in the elderly group(P<0.05),Sirt1 expression in the elderly treatment group was higher than the elderly group(P>0.05),Sirt3 expression in the elderly treatment group was higher than the elderly group(P>0.05);apoptosis-related protein Caspase3 and Bax gene expression in the elderly group was older than the elderly treatment group Elevated(P<0.01),the Bcl-2 expression in the elderly group was lower than that in the elderly treatment group(P<0.05);by sequencing and analysing the m RNA transcriptome in the thymus tissue,it was found that the expression characteristics of the tissue transcriptome compared with the elderly group The tendency of young rhesus monkey thymus expression characteristics,a total of 312differentially expressed genes related to thymus tissue aging were detected,including305 genes up-regulated with age and 7 genes down-regulated with age;3D PCA analysis found that Compared with the elderly group,the overall transcriptome characteristics of the elderly treatment group had a clear tendency toward the direction of the young group;cluster analysis and trajectory analysis found that the expression characteristics of the thymus after BMSC transplantation were more inclined to the expression characteristics of the thymus in the young group.Through GO enrichment analysis,it was found that the down-regulated genes after treatment with up-regulation of aging were mainly enriched in cytokine and cytokine receptor interaction,renal tubular formation,synaptic signal transmission,positive regulation of ion transport,and osteoclast differentiation regulation,etc.on the pathway.Conclusion:1.An effective evaluation method for thymus function reconstruction of natural aging macaques is determined;2.Establish a preparation method for macaque BMSCs to obtain BMSCs whose cell morphology,growth characteristics,cell surface antigens and differentiation potential meet the standards;3.BMSC transplantation in juvenile rhesus monkeys can promote the reconstruction of the thymus structure of aging cynomolgus monkeys,improve the T cell output ability and thymus hormone production function.4.Transplantation of BMSCs in young rhesus monkeys regulates genes related to aging and apoptosis,and improves thymus degeneration. |
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