| Objective:The treatment of the Bone Marrow Mesenchymal Stem Cells(BMMSC)was implemented for the elderly macaques and serum proteomics database was obtained by using the Data Independent Acquisition(DIA)analysis of digitized biological samples based on mass spectrometry to select the differential proteins of the different ages macaques and senile macaques after BMMSC treatment,so the change rule of the serum protein composition and content of the elderly macaques after the treatment of the BMMSC and macaques under differernt ages can be revealed.While by comparing and analyzing the biological functions and signaling pathways of different proteins,we should find the key regulators of the elderly macaques after BMMSC treatment and look for evaluation indexes of therapeutic effects.Methods:1.Evaluation of the natural aging macaque modelsThe elderly group consisted of 6 female monkeys aged 23-25 years old.Based on the research of heart,lung,kidney,brain and other structural and functional degenerations in this group,we focused on the observation of the phenotypes of elderly monkeys such as the morphological features of the aortic arch after HE staining,while observing the facial features of elderly macaques,the number of hair colors.2.Preparation and identification of BMMSCThe infant macaques were intramuscularly injected with 3%pentobarbital(1 mg/kg),subsequently the bone marrow was harvested by bone marrow aspiration under the sterile conditions.The BMMSC primary cells were cultured by whole bone marrow tissue adherence,purified and expanded to the P4 generation for experiments.The morphological characteristics of P4 generation BMMSCs were observed under the microscope;the expression levels of BMMSC surface markers CD29,CD34,CD90,CD 105 were analyzed by flow cytometry;BMMSCs were subjected to differentiation experiments which contain the function induction of adipogenesis,osteogenesis and cartilage.3.The grouping and treatment of the macaquesThe experimental animals were divided into 4 groups:3 in the infant group,3 in the young group,4 in the elderly group,and 4 in the treatment group,all of which were female.BMMSCs were injected via femoral vein at 1×107 cells/kg,infused once every other day,3 consecutive treatments,venous blood was taken for blood lipid creatinine and other tests at 30 days after treatment,and venous blood was taken and tested for blood creatinine at 180 days after treatment and followed an autopsy.4.Detection of blood lipid and creatinine and the HE stainingin of aortic arch of rhesus monkeyVacuum blood sampling method was used to take the elderly group's and 30 days after treatment,180 days of macaque femoral vein blood which was after treatment for the blood lipid detection;When 180 days after the treatment of BMMSC,the macaques were killed by intramuscular injection of 3%excess pentobarbital excess,and the aortic arch of the treatment group and the elder control group were stained with HE to observe the morphological and structural characteristics.5.DIA proteomics analysisThe whole blood of the macaque femoral vein was centrifuged by vacuum blood collection to obtain serum and transported to Shanghai Jingtian Company for DIA Proteomics analysis which was used to observe the expression of serum proteins in each group.The proteomics quantitative technique was used to screen out the serum differential proteins of aged cynomolgus monkeys of different ages and after BMMSC treatment,then the Go(Gene Ontology)functional analysis,KEGG(Kyoto Encyclopedia of Genes and Genomes)database pathway analysis and other analysis were used screen out the most meaningful evaluation indicators of efficacy.Results:1.Model evaluationIn this experiment,four natural aging macaque models aged 23-25 were successfully screened.The research team conducted simultaneous PET-CT whole-body imaging scans,and HE staining,Masson staining,and Tunel staining of the lungs,thymus,and heart,etc,and observed that the structural and functional degeneration results of all organs are consistent with the phenotype of natural aging macaques;In this experiment,erythema on the face of elderly macaques was observed,the number of hairs around the face and back was reduced,the hairs were dull,and the light color was reduced;HE staining of the aortic arch showed obvious massive blue staining and obvious calcification,which was consistent with the phenotype of aging monkeys.2.BMMSC cultivation and identificationMicroscopic observation of P4 generation BMMSC showed a fusiform spiral shape with uniform size;osteogenic induction with alizarin red staining was positive,adipogenic induction with oil red O staining was positive,and cartilage induction was induced by Alslan staining observed positive under the microscope.Through the flow analysis of P4 generation BMMSC surface markers CD29,CD34,CD90,CD105,the positive expression rates were 97.3%,1.5%,98.4%,99.8%.3.Determination of blood lipid and creatinine and HE staining of aortic arch in elderly monkeysAfter 30 days and 180 days of BMMSC treatment of aging macaques,reducing the low density lipoprotein cholesterol(LDL-C,low density lipoprotein-cholesterol),triglyceride(TG,Triglyceride)/high density lipoprotein cholesterol(HDL-C,high density lipoprotein)-cholesterol),creatinine level,increasing the HDL-C level,suggesting that BMMSC can improve the blood lipid level of elderly cynomolgus monkeys;HE staining of the aortic arch 180 days after treatment showed no pathological phenomena such as calcium salt deposition and cell necrosis.4.DIA proteomics analysis1006 kinds of proteins were detected of the 14 serum samples,the standard for screening differential proteins is p-value<0.05,The absolute value of Fold change>1.5.44 differential proteins were detected in group D and C,including 11 up-regulated differential proteins and 33 differential down-regulated proteins.There are the up-regulation of differential proteins such as COMP(cartilage oligomeric matrix protein)which was associated with vascular aging,TUBA 1A(Tubulin a chain)which was associated with neuronal aging,and NKAP(NF-?-B-activating protein)which was related to the hematopoiesis,there are the down-regulation of differential proteins such as S100 related to the neuronal aging,LCN2(Lipocalin 2)which was associated with cardiovascular disease,and CSF1R(Colony stimulating factor 1 receptor)and CORO1C(Coronin)which were associated with tumor.Compared with group A,group D were screed 83 kinds of differential proteins,including 43 up-regulated differential proteins and 40 down-regulated differential proteins.There were the up-regulated differential proteins such as TBC1D2(TBC1 domain family member 2)which was associated with metabolism,SPATA18(Spermatogenesis associated 18)which was associated with the reproductive function etc,while the down-regulation of differential proteins such as COMP,POSTN(Periostin)which was associated with inflammation etc;Compared with group A,group B were screened 80 differential proteins,including 38 up-regulated differential proteins and 42 down-regulated differential proteins.There were the up-regulated differential proteins such as RSU1(Ras suppressor protein 1)which was associated with tumor,5'-3'exoribonuclease 1 which was related to neuronal aging etc,while the down-regulation of differential proteins such as COMP,POSTN etc.Compared with group A,group C were screened 90 differential proteins,including 52 up-regulated differential proteins and 38 down-regulated differential proteins.There were the up-regulated differential proteins such as G6PI(Glucose-6-phosphate isomerase)which was associated with Neuron aging,CHI3L1(Chitinase 3 like 1)which was associated with immune function,while the down-regulation of differential proteins such as POSTN,COMP,IGL?10-54(Immunoglobulin lambda variable 10-54)which was related to immune function etc.Conclusions:1.The research team successfully screened 4 cynomolgus monkeys aged 23-25 years old as the natural aging model.Through imaging PET-CT and histopathological analysis,it was found that the thymus gland of the cynomolgus monkeys was atrophy,more fat was filled,and inflammatory cells appeared in the lungs and increased myocardial fibrosis,etc;this experimental study found that aging cynomolgus monkeys showed dull hair,erythema on the face,and localized calcification of the aortic arch.2.The whole bone marrow adherent culture method was used to isolate and culture the P4 generation BMMSC,which showed a fusiform spiral shape,uniform size,positive expression of CD29,CD90,CD 105,negative expression of CD34,high purity,and the potential of adipogenic and chondrogenic differentiation.3.After 30 days and 180 days of BMMSC treatment in elderly macaques,the expression levels of LDL-C,TG/HDL-C,and creatinine in the treatment group were significantly reduced,and the expression levels of HDL-C were increased.The LDL-C,TG/HDL-C,and HDL-C may be used as indicators for evaluating the efficacy of BMMSC transplantation in the treatment of elderly rhesus monkeys.4.After BMMSC treatment of elderly cynomolgus monkeys,the characteristics of aging-related serum proteome were reversed,and the protein expression of the treatment group was inclined to the infant group and the young group;compared with the elderly group,the expression of S100 in serum was significantly reduced after BMMSC treatment(Fold change=-20.55,p value<0.05);COMP expression has been decreasing in the young and old stages,and the expression level increased after BMMSC treatment and approaching the early stage;compared with the treatment group,NKAP was not detected in the elderly group;compared with the elderly Compared with the treatment group,LCN2,CSF1R,CORO1C,CSTB were not detected in the treatment group;the expression of RSU-1 has been increasing from childhood to youth to old age,and the protein was not detected in serum samples after BMMSC treatment,the above results It shows that the COMP,LCN2,S100,NKAP,CSF1R,CORO1C,etc.may be used as alternative markers for BMMSC treatment of aging and aging-related diseases such as cardiovascular disease and tumor. |
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