Changes Of Spleen B Lymphocytes In D-galactose Induced Subacute Aging Mice And The Anti-aging Effect Of Yunke (⁹⁹Tc-MDP)

Aging is a process of the gradual decline of physiological functions anddegenerative changing of tissues and organs, which ends in death. There are a lot oftheories about aging, representative ones are Free Radical Theory, Immunity Theory,Mitochondria Theory, and Telomere Theory. Among them, the Immunity Theory wasput forward by Wolford in the60s of the20thcenturies. It suggested that the decline ofimmune functions is an important factor that causes aging, and immune systemessentially determines the body’s aging.Spleen is the body’s vital organ for peripheral immunity. It is the place whereimmune cells live and immune responses are triggered, and B cells account for about60%of the total number of splenic lymphocytes. B lymphocytes are the only cells in thebody that produce antibodies, meanwhile they are antigen presenting cells. They play amain role in the humoral immunity and cellular immunity in the body. During aging, thenumber of B cells drops, the gene expression maps of immune globulin and thefrequency of somatic protein-altering mutations change, leading to the body’s immunesystem function disorder.YunKe(⁹⁹Tc-MDP) is Technetium[⁹⁹Tc] methylene diphosphonates injection, usedto treat ankylosing spondylitis, rheumatoid arthritis, hyperthyroidism with invasiveprominent eyes etc. currently in clinical.⁹⁹Tc can easily gain and lose electronics so asto constantly clear free radicals in the body, preserve the vitality of superoxidedismutase（SOD）, and inhibit the generation of pathological complex.This thesis uses D-galactose to establish subacute aging mice model, and on thebasis of biology identification carry out preliminarily discussion and analysis about thechanges of spleen B cells, Macrophages, dendritic cells of aging mice, in order toprovide experimental basis of further study of the relationship between the immunesystem and aging, and to research the reversal effect of YunKe on aging mice, so as to offer theory basis for its use as an anti-aging drug in clinical.Part Ⅰ The Establishment and Biology Identification ofD-galactose Induced Aging MiceObjective: Establishing subacute aging mice model with D-galactose.Methods:16healthy Kunming mice of two months old were divided into twogroups at random. The aging model group were injected with100g/L D-galactose,0.25ml/20g, into their back necks. The control group were injected with the same dosesof saline. The animals’ living conditions and biological behaviors were observedeveryday and the dynamic changes of their weights were measured regularly. After42days, the eyes of mice were picked for blood sampling, then serum was taken throughcentrifuging, and the SOD viability and malondialdehyde（MDA） concentration weremeasured.Results:42days after being injected with D-galactose, compared with the controlgroup, the mice of the model group showed listlessness, consumption reducing,withered and darkened skin, and slow weight growth. The results of biologyidentification showed that SOD viability was2.16±0.43mkat/L, and MDAconcentration was4.14±0.82μmol/L in the serum of the model group. Compared withthe control group5.52±1.55mkat/L,1.24±0.21μmol/L, the SOD viability fell, and MDAconcentration increased（P＜0.01）.Conclusion: D-galactose induced aging mice model has been successfullyestablished, providing an experimental object for the study of the changes of Bcells,Macrophages,dendritic cells of aging mice and the anti-aging effects of Yunke.PartⅡ TheActivation-and Proliferation-and Memory-relatedChanges of B cells of D-galactose Induced Aging MiceObjective: Analyzing the changes of B cells,Macrophages,dendritic cells andcytokine in serum and their significance of the D-galactose induced aging mice, in orderto lay the theoretical foundation for the further study of the aging mechanism.Methods: Healthy Kunming mice of two months old were injected withD-galactose to establish aging mice model, and then taken biological identification. The analysis of spleen index was carried out with weighing method; the analysis of themajor membrane type molecules of spleen B cells, macrophages and dendritic cellswere carried out with immune fluorescence and flow cytometry; the analysis oftransformation stimulate index of spleen B cells was carried out with MTT; the analysisof spleen structure was carried out with hematoxylin-eosin staining; the analysis of theFas expression was carried out with immunohistochemistry; the analysis of theconcentration of IL-4and IFN-γ in serum were carried out with ELISA.Results: The spleen index of model mice group was2.75±0.86, and compared withthe control group’s5.15±2.12, it decreased（P＜0.05）; the percentages of CD20⁺, CD40⁺,CD86⁺/CD20⁺CD86⁺, CD86⁺/CD40⁺CD86⁺of spleen B cells,CD11c⁺/MHC-Ⅱ⁺CD11c⁺of dendritic cells of the model group were56.82±2.58,43.60±3.60,2.25±0.15,4.38±0.42,5.34±0.32, and compared respectively with thecontrol group’s71.76±3.77,56.66±4.91,3.36±0.29,7.70±0.88,7.88±0.77, they decreasedobviously（P＜0.01）; the percentages of CD196⁺/CD20⁺CD196⁺,CD196⁺/CD40⁺CD196⁺of spleen B cells of the model group were10.68±0.81,10.98±1.88, and comparedrespectively with the control group’s9.22±0.80,5.68±1.28, they increased obviously（P＜0.01）; the percentages of CD45RA⁺/CD20⁺CD45RA⁺,CD45RA⁺/CD40⁺CD45RA⁺ofspleen B cells and CD11b⁺/MHC-Ⅱ⁺CD11b⁺of macrophages of the model group were14.04±1.35,35.40±2.24,9.78±0.63,and compared respectively with the control group’s15.36±1.02,39.20±2.93,10.76±0.86, they decreased（P＜0.05）; the SI of the model groupwere1.28±0.15（24h）,1.55±0.10（48h）, and compared respectively with the controlgroup’s1.60±0.21,2.63±0.19, they decreased obviously（P＜0.01）; the SI of model micegroup was1.19±0.17（72h）, and compared with the control group’s1.40±0.08, itdecreased（P＜0.05）; the result of hematoxylin-eosin staining showed that the white pithof spleen of the model mice group was narrowed, cells reduced, red pulp expanded, theboundary of the white pith and the red pulp unclear, the fringe area unobvious; the Fasexpression of spleens of the model group increased, the average level of IL-4and IFN-γin serum of the model group were8.28±0.46,82.93±2.74ng/L, and comparedrespectively with the control group’s12.49±0.53,125.41±3.16, they decreasedobviously（P＜0.01）.Conclusion: In the body’s aging process, the spleen shrinks gradually, the white pith narrows, the red pulp expands, the boundary of the white pith and the red pulpbecomes unclear, the expressions of the activation-and memory-related majormembrane type molecules of spleen B cells change，activated cells decrease, memorycells increase, the activation degree of antigen presenting cells decrease, and theexpressions of IL-4and IFN-γ in serum decrease, the body’s reactiveness to foreignantigen drops, and the Fas expression in the spleen falls gradually, cell apoptosisincreases, resulting in the function disorder of the immune system.Part Ⅲ The Anti-aging Effect of Yunke(⁹⁹Tc-MDP)Objective: Researching the anti-aging role of⁹⁹Tc-MDP on D-galactose inducedaging mice.Methods:30healthy Kunming mice of two months old were divided into sixgroups at random: the control group, the model group, the positive control group, thelow dose group, the middle dose group, and the high dose group. The aging modelgroup were injected with100g/L D-galactose（0.25ml/20g） into their back necks,20minutes later, intragastric infusions with0.85%physiological saline（0.2ml/10g） wereperformed, for42days consecutively; the control group were injected with same dosesof physiological saline,20minutes later, intragastric infusions with0.85%physiologicalsaline（0.2ml/10g） were performed, for42days consecutively; the positive controlgroup were injected with100g/L D-galactose（0.25ml/20g）, into their back necks,20minutes later, intragastric infusions with Vitamin E（0.2ml/10g） were performed, for42days consecutively; the⁹⁹Tc-MDP groups were injected with100g/LD-galactose（0.25ml/20g） into their back necks for42days consecutively,20minuteslater, they were injected with corresponding doses of⁹⁹Tc-MDP:3.5×10-4μg/20g forthe low dose group,7.0×10-4μg/20g for the middle dose group,1.4×10-3μg/20g forthe high dose group, administered once every other day. The animals’ living conditionsand biological behaviors were observed everyday and the dynamic changes of theirweights were measured regularly. After42days, eyes of the mouse were picked forblood sampling. The analysis of the spleen index was carried out with the weighingmethod; the analysis of the importmant membrane type molecules of spleen B cells wascarried out with immune fluorescence and flow cytometry; the analyses of theconcentration of IL-4and IFN-γ in serum were carried out with ELISA. Results: the mice of the model group showed listlessness, consumption reducing,withered and darkened skin, slow weight growth, inactive and easy to catch, slightlyuplift backs, showing obvious signs of aging. And the Yunke group showed similarconditions with the normal control group, with no obvious signs of aging; the spleenindex declined in the model group, while increased in the positive control group and theYunke group, and the degrees of spleen atrophy of the latter two groups decreased; thepercentages of CD20⁺, CD40⁺, CD45⁺of spleen B cells decreased in the model groupwhile compared to the control group, yet they increased in the positive control groupand the Yunke group; the average level of IL-4and IFN-γ in serum dropped in themodel group, yet increased in the positive control group and the Yunke group.Conclusion: Yunke can improve the immune function, it has an anti-aging effect.