Age-related Changes Of Spleen Extracellular Matrix And Splenic B Lymphocytes And Effect Of Allogeneic Bone Marrow-derived Mesenchymal Stem Cell Transplantations On These Changes In Mice

Objective1. To investigate the age-related changes in the spleen extracellular matrix (ECM) of BALB/c mice, and explore the involvements of (3-catenin signaling in the senescence of spleen ECM.2. To observe the age-related changes in the frequency of splenic B lymphocytes, proportions of specific B cell subsets, as well as relative mRNA expression levels of proliferation, apoptosis, and cell cycle-related genes, and to explore the impact of spleen ECM senescence on B cells of BALB/c mice.3. To explorer the effect of allogeneic transplantations of bone marrow-derived mesenchymal stem cells (BMSCs) isolated from C57BL/6J mice on the senescence of the spleen ECM and B lymphocytes of BALB/c mice in vivo.Methods1. Female BALB/c mice were divided into three groups according to age:young adult (2-4 months), middle aged (8-10 months) and old (18-20 months). Paraffin embedded spleen sections were stained with hematoxylin and eosin (H&E) for evaluation of morphology. Types and spatial distributions of collagens were detected using the sirus red staining. Protein expressions of collagen 1, elastin, and fibronectin were detected by immunohistochemistry.2. Spleen samples from different age groups were harvested, then quantitative (real-time) PCR was performed to evaluate the age-related changes in mRNA expression levels of ECM compoments including agrin, collagen Iα1, collagen Ⅲα1, collagen IVal, collagen VIα1, elastin, fibrillin 1, fibronectin 1, fibulin 1, laminin a5, and MMP-2, and the mRNA expression levels of β-catenin and its upstream regulators DVL2, DVL3, CK1, and GSK-3β, downstream transcriptor TCF4, and target genes PPARδ, Axin2, c-myc, and cyclin D1, as well as B cell specific marker CD 19. Western blot was performed to investigate the protein expression levels of β-catenin and CK1.3. Splenic mononuclear cells were prepared and stained with fluorescence labelled antibodies. Then flow cytometry was performed to determine the frequency of CD19+ B lymphocytes, as well as those of the CD19+CD5+, CD19+CD24+, CD19+IgM+IgD-, and CD19+IgD+B cell subsets of mice in different age groups.4. Splenic B lymphocytes were separated and collected by flow cytometry. Real-time PCR was performed to evaluate the age-related changes in mRNA expression levels of Bax, Bcl-2, caspase 3, caspase 8, cyclin D1, cyclin E1, Ki-67, p16INK4a, CD5, and CD24, as well as the mRNA expression levels of β-catenin and its upstream regulators DVL2, DVL3, CK1, and GSK-3β, downstream transcriptor TCF4, and target genes PPARδ, Axin2, and c-myc of splenic B cells.5. BMSCs were isolated from femurs and tibias of C57BL/6J mice and subcultured to the 6th passage until animal administration.6. Each mouse in the administration groups was infused in the tail vein biweekly with a final concentration of 1×106 BMSCs suspended in 100 μl of PBS, for a total of four injections. Two weeks after the final injection, animals were sacrificed and spleens were removed. Then experimental procedures mentioned above were repeated and results were compared with naive control groups.ResultsPartⅠ1. The struture of the spleen of old BALB/c mice were disorder with more deposition of hemosiderin and collagen I compared with young mice.2. The mRNA expression levels of agrin, collagen Ial, collagen Ⅲα1, collagen IVα1, elastin, and fibrillin 1 of the spleen were lower in old mice than in young groups (P<0.01 or P<0.05). No statistical differences were detected in the mRNA expression levels of collagen VIα1, fibronectin 1, fibulin 1, laminin α5, or MMP-2 among different age groups.3. The mRNA and protein expression levels of β-catenin and CK1 of the spleen were significantly lower in old mice compared with youg mice (P<0.05 or P<0.01), the mRNA expression levels of PPARδ, cyclin D1, and CD44 of old mice were lower than youg mice (P<0.01 or P<0.05); the mean valus of DVL2, DVL3, GSK-3β, TCF4, and Axin2 mRNA expression levels of old mice were lower than young mice, yet not stastically significantly.4. After 4 times of allogenic BMSC transplantations, the old mice exhibited upregulated mRNA expression level of collagen Ⅲα1 (P<0.01), downregulated levels of collagen IVal and fibronectin 1 in the spleen (P<0.05 or P<0.01). The mRNA expression levels of TCF4 and CD44 in the spleen of old mice were also downregulated (P<0.05). The mRNA expression levels of other ECM components or molecules involved in P-catenin signaling were not affected.Part Ⅱ1. The frequency of splenic CD 19+B lymphocytes was maintained during aging. The old mice exhibited higher proportions of CD19+CD5+and CD19+IgD+B cell subsets (both P<0.01), and lower proportions of CD19+CD24+and CD19+IgM+IgD-B cell subsets (both P<0.01) compared with young mice. The mRNA expression levels of Ki-67, and CD24 of splenic B cells were lower in old mice (both P<0.01), Bcl-2, caspase 8, pl6INK4a, and CD5 were higher (P<0.05 or P<0.01), and Bax, caspase 3, cyclin D1, and cyclin E1 were even with young mice.2. No significant differences were observed in the mRNA expression levels of β-catenin, DVL2, DVL3, CK1, PPARδ, CD44, or Axin2 in splenic B cells among different age groups.3. After four times of allogenic BMSC transplantations, the proportion of B cells as well as the mRNA expression level of CD 19 in the spleen of old mice were both increased (both P<0.01). The old mice also exhibited diminished proportions of CD19+CD5+, CD19+CD24+, and CD19+IgD+B cell subsets (P<0.05 or P<0.01), as well as upregulated mRNA expression levels of Ki-67, and p16INK4a (both P<0.01), and downregulated levels of CD5, CD24, and caspase 8 (P<0.05 or P<0.01) in splenic B cells compared with young mice.Conclusion1. The old mice exhibited decresed mRNA expression levels of extracellular matrix compoments in the spleen.2. The β-catenin signaling is essential for the senescence of spleen ECM.3. Splenic B lymphocytes are maintained in frequency with advanced age, but experience lower proliferation, higher apoptosis, and restricted cell cycles. These age-related changes might be related with the spleen ECM aging.4. Allogeneic BMSC transplantations may influence the age-related changes in the mRNA expression levels of ECM compoments and P-catenin signaling in the spleen of old BALB/c mice. BMSC transplantations may also alter the frequency, specific subsets proportions, and mRNA expression levels of proliferation or apoptosis-related genes of splenic B cells.