Abstract: BACKGROUND: Spleen has the functions of blood storage, hematopoiesis, and immunity. With the increase of age, the structural degeneration and functional decline of spleen lead to the impairment of immune system function, thus accelerating the aging process of the body. The treatment of spleen aging in tree shrews with highly active umbilical cord mesenchymal stem cells has not been reported. 
OBJECTIVE: To explore the intervention effect and mechanism of highly active umbilical cord mesenchymal stem cells on spleen aging in tree shrews. 
METHODS: Highly active umbilical cord mesenchymal stem cells were isolated, cultured, and obtained from the umbilical cord tissue of newborn tree shrews by caesarean section. The differentiation abilities of adipogenesis, osteogenesis, and chondrogenesis were detected by three-line differentiation kit. Cell cycle and surface markers were detected by flow cytometry. The second generation of highly active umbilical cord mesenchymal stem cells were transfected with Genechem Green Fluorescent Protein with infection complex values of 100, 120, 140, 160, 180, and 200, respectively, to screen the best transfection conditions. After transfection, the fourth generation of highly active umbilical cord mesenchymal stem cells was injected into the tail vein of tree shrews in the elderly treatment group. The young control group and the aged model group were not given special treatment. After 4 months of treatment, the spleen tissue was taken and the structure of the spleen was observed by hematoxylin-eosin staining. β-Galactosidase staining was used to detect the activity of aging-related galactosidase. Immunohistochemical staining was used to detect the expression levels of p21 and p53 proteins. Ki67 and PCNA immunofluorescence staining was used to detect cell proliferation activity. Immunofluorescence staining was used to detect the expression levels of spleen autophagy protein molecules Beclin 1 and APG5L/ATG5. Reactive oxygen species fluorescence staining was used to detect the content of reactive oxygen species in spleen tissue. CD3 immunofluorescence staining was used to detect the change of the proportion of total T lymphocytes. The secretion levels of interleukin 1β and transforming growth factor β1 in spleen were detected by enzyme linked immunosorbent assay. The distribution of highly active umbilical cord mesenchymal stem cells labeled with green fluorescent protein in spleen tissue was observed by DAPI double staining of nucleus. 
RESULTS AND CONCLUSION: (1) Highly active umbilical cord mesenchymal stem cells grew in a short spindle shape with fish-like growth, with a large proportion of G0/G1 phase, and had the potential to differentiate into adipogenesis, osteogenesis, and chondrogenesis. (2) Multiplicity of infection=140 and transfection for 72 hours were the best conditions for labeling tree shrews highly active umbilical cord mesenchymal stem cells with Genechem Green Fluorescent Protein. (3) Compared with the aged model group, in the aged treatment group, the spleen tissue cells of tree shrews were arranged closely, and the area of white pulp was increased (P < 0.01); the boundary between red pulp and white pulp was clear; the proportion of germinal centers did not show statistically significant difference (P > 0.05). The activity level of galactosidase related to spleen tissue aging was decreased (P < 0.001), and the expression levels of aging protein molecules p21 and p53 were down-regulated (P < 0.001). The expression levels of proliferation-related molecules Ki67 and PCNA were up-regulated (P < 0.001, P < 0.05); expression levels of autophagy-related molecules Beclin 1 and APG5L/ATG5 were up-regulated (P < 0.001), and the content of reactive oxygen species decreased (P < 0.001), and the proportion of CD3+ T cells increased (P < 0.05). The secretion level of interleukin 1β in the aging-related secretion phenotype decreased (P < 0.001); no significant difference was found in transforming growth factor β1 level (P > 0.05). Compared with the young control group, the above indexes were significantly different in the elderly treatment group (P < 0.05). (4) Green fluorescent cells labeled with green fluorescent protein were observed in spleen tissue of tree shrews the elderly treatment group by frozen tissue section observation. The results show that intravenous infusion of highly active umbilical cord mesenchymal stem cells can migrate to spleen tissue, inhibit the production of reactive oxygen species, down-regulate the expression of aging-related proteins, induce autophagy, promote cell proliferation, reduce chronic inflammation, and then improve the structure and function of spleen tissue. 

Key words: highly active umbilical cord mesenchymal stem cell, tree shrew, spleen, aging, immune, autophagy, inflammation