The Reversal Effect Of Mesenchymal Stem Cells On The Aging-rats

BACKGROUND&OBJECTIVEChina is rapidly into aging society, China's current 60 or older is about 1.69 billion. It is forecast that by 2050 China will account for over 60 years old, reaches 30% variety of aging 31%, bring more serious trouble and problems.It's unavoidable to be aging and death for any species of individuals but so far far our understanding of the mechanism of aging is still quite limited. Given its importance, has been the focus of biological research.After years of research, now about aging mechanisms have proposed many theories, each theory has its advantages and disadvantages. Such as radical theory, genetic programming theory, error catastrophe theory, crosslinking theory, lipofuscin cumulative theory, endocrine function decline theory, and so on, are discussed from different angles of mechanism and countermeasure aging happen. Nearly 50 years of theoretical and experimental proof, radical doctrine is one of the most persuasive aging theory.Currently considered, the augmented peroxidation caused by D-galactose metabolic disorder is important factors to aging reaction. By continuous injection D-galactose, because its metabolites galactose can't further metabolism and piling up in cells, it will cause cell swelling, and cause animal tissues and cells degenerate and appeare functional changes. Meanwhile D-galactose produces more free radicals in the body, scavenging ability of the body, which triggers the chain reaction of lipid peroxidation, produce large amounts of lipid peroxide of MDA, increasing the damage to cells. D-galactose animal model method is simple, inexpensive and stable, has become a nationally recognized animal model of aging. So subacute aging model induced by D-galactose is adopted in this experiment.Mesenchymal stem cells are derived from the early development of embryos mesoderm multipotent, and MSCs is a body of connective tissue cells. Mesenchymal stem cells can differentiate into osteogenic cells, fat cells,cartilage cells, muscle cells and so on at the appropriate condition in vitro. And MSCs can also differentiate into neurons, glial cells, liver cells, endothelial cells through endoderm. With the study of MSCs, it has not any multilineage differentiation potential but also secrete various neurotrophic factors including brain-derived neurotrophic factor, nerve growth factor, vascular endothelia growth factor, vascular endothelial growth factor receptor2, basic fibroblast growth factor, and so on. These factors played a role of treatment in injury such as promote angiogenesis, tissue regeneration.Based on the researching regarding to senescence recentent years, it is considered to be a disease related to gene. And some genes or cells have already been confirmed to closely related with senescence,which could be summarized as cell cycle controlling gene, TLMA controlling gene, Growth inhibiting protein gene, DNA/protein synthesis and restoration gene. Aging is also concerned with the expression and activity of of transcription factors (transacting factors). Aging relevant genes which Have cell proliferation regulation function, including P21,P16,cyclin D1,cyclin-dependent kinase 2(CDK2),P53,Rb,Proliferating Cell Nuclear Antigen,PCNA, and so on. Linkages between these genes may constitute a cell senescence gene regulation chain. Therefore, this issue will be discussedwhether MSC affect expression of p16, p21, proliferating cell nuclear antigen, which may be the role of mesenchymal stem cells play in the mechanism of aging.To sum up, our research to mesenchymal stem cells in the anti-aging. This research will provide mesenchymal stem cells play anti-aging based provide theory and practice. If can obtain the desired results, after a good design research, the basis of its application in clinical time not too long, wide prospect.In summary, we intend to study the anti-aging effect of mesenchymal stem cells, and this research will provide theoretical and practical basis about MSCs in anti-aging effects. If the expected results, the basis of well-designed study, its clinical application will not take too long and bright future.METHODSPart I:Mesenchymal stem cells were cultured in vitro and identifiedRat bone marrow mesenchymal stem cells were isolated and purified by adherent culture. After the tibias and femurs were dissected from rat, the marrow was flushed out with PBS buffer under aseptic condition. The cell suspension were inoculated into L-DMEM medium containing 10% fetal bovine serum. Cells were not digested until the cells reached confluence over 80% after medium changes. P3 MSC populations were collected and examined as follow:①The morphologic changes of MSCs were detected by inverted light microscope and HE dyeing;②Growth curve and clone formation was measured by MTT and experiment of clone formation;③Detecting expressed the surface marker expression and the growing circle of BMSCs by flow cytometric analysis; ④The efficiency of differentiation into osteoblast was assessed by related methods. Part II:Establishment of aging model, study home of MSCs in aging rats.①Subcutaneous injection of D-galactose, consecutive 4 months, establish rat model of aging. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were measured wtih hydroxylamine and chromatometry respectively.②MSCs were isolated from aged and young SD rats. Cell morphology and growth status were observed by a inverted microscope, expression of surface antigen was detected by flow cytometry, the fibroblast colony-forming unit (CFU-F) were compared between two groups. Electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups.③MSCs were isolated and purified from femur in the rat by adherent culture, the third generation MSCs were infected by GFP-adenovirus carrier. The cell morphological changes were observed under the inverted phase contrast microscope, and the expressi on of GFP was observed under the fluorescence microscope to conf irm success of the labeling of MSCs. we can draw the cell growth curve by MTT. Furthermore, the cell cycle and the surfacemarkers (CD29, CD34, CD44, CD45) of the labeled MSCs were detected through flow cytometry and were compared with those of non-labeled MSCs.④After infected by GFP, MSCs are differentiated into cell suspension with density of 3×107/L. Then 3×106MSCs was transplanted into the ventricle of rats and the expressi on of GFP in the rat was examined under fluorescentmicroscope.PartⅢ:The study of MSCs'effect on the aging ratsHealthy SD rats were randomly divided into three groups at random:normal control group, aging model group and MSCs group. The aging model group were injected with D-galactose for 4 months to establish the aging rats. ①After the end of experiment, rats were executed and get rat's serum. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were measured wtih hydroxylamine and chromatometry respectively. To detect serum calcium, alkaline phosphatase(ALP), estradiol(E2),progesterone(P), luteinzing hormone(LH,follicle stimulating blood hormone(FSH) and testosterone, using the automatic biochemical analyzer and microplate reader analyzer.②The difference of three groups in organizational structure were observed by conventional paraffin sections. We study the role of MSCs in anti-aging from the level of functional, biochemical and cellular.③To measure the thymus index and spleen index with weighing method; The spleen lymphocyte proliferation activity were measured by MTT; the level of IL-2, IL-10 and IL-7 were measured by ELISA; thymus CD4 + CD8 + T cells were detected with Flow cytometric analysis; the thymus organizational structure is examined by transmission electron microscopy.④Bone density scans to measure bone mineral density of rats (BMD); the trabeculae was observed under scanning electron microscopy.Part IV:The mechanism of MSCs'treatment on the aging ratsAfter the end of experiment, rats were executed. To measure the expression of P16, P21, Proliferating Cell Nuclear Antigen in rats'organizationStatistical methods:The result of experiment data with mean±standard deviation (x±S),that significant for the 0.05 standard, using SPSS 17.0 statistical packages for statistical analysis. Group compared with one-way ANOVA; two-two comparison with LSD when homogeneity of variance, with Dunnett's T3 and Dunnett's C method when Heterogeneity of variance; the main effect of each factor and interaction effects with Factorial analysis of variance; the overall mean differences of two independent sample with Independent-samples T test; repeat measurements analysis of variance with repeated measures.ResultPartⅠ:Mesenchymal stem cells were cultured in vitro and identifiedRat bone marrow-derived MSCs were isolated by adherent culture. After passage, the cells were spindle-shaped, uniform. P3 cells were inoculated in low-density to the culture plate, and the scattered clones were formated after 1 week. MSCs cells were into index growth 3 days after inoculation, and into platform period from the fifth day. G0G1 phase of MSCs cells is about 80%, using Flow cytometry. MSCs cells are uniformly expressing CD29, CD44, and weakly expressing CD34, CD45. Cells were successfully induced into osteoblasts, adipocytes cells.PartⅡ:The study of home of MSCs in aging rats.①Compared with the control group, the activity of SOD was significantly lower, the content of MDA increased significantly in aging model group (P＜0.05)②Compared with control group, the growth velocity was obviously slowlier, and the CFU-F of the primary BMSCs was lower in aging model group (P＜0.05). The mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic, using electronic microscope.③The expression of GFP began at 24 hours af ter t ransfection, and reached the peak at 72 hours and decreased in 1 week; however, the transient expression remained for more than 4 weeks. The morphological feature of the uninfected MSCs was similar to that of the MSCs-GFP, and there were no significant differences in the cytoactive and cell cycle between the MSCs and the MSCs-GFP (P> 0.05). ④MSCs can be found in the thymus,spleen,ovaries and testis of senile rats. Furthermore, the injected labeled cells could still be detected through pathology after 7 days.PartⅢ:The study of MSCs' effect on the aging rats①Compared with the aging model group, the activity of SOD increased significantly, the content of MDA was significantly lower in MSCs group, and there were significant differences (P<0.05). Microplate reader analyzer detection of serum hormone levels showed that levels of E2,P and T were higher, and the level of LH,FSH were lower in MSCs group, compared with aging model group (P<0.05) Automatic biochemical analyzer detection showed that the content of serum calcium and serum ALP significantly raised in the model group comparing with normal control (P＜0.05); he content of serum calcium and serum ALP significantly decreased in the MSCs group comparing with model groups (P＜0.05)②The tissues injury is serious in aging model group rats; and the tissues pathological injury had obvious repaired in MSCs group, compared with aging model group. To detect the proportion of different atrophic seminiferous tubules in testis at 100 times light microscope. The result showed the proportion of atrophic seminiferous tubules decreased, and the the proportion of normal seminiferous tubules raised in MSCs group, compared with the model group (P＜0.05).And the number of corpus luteum and ovarian follicle rised in MSCs group, compared with the model group (P＜0.05), the difference was statistically significant.③MSCs can enhance immune function of aging rats, apperaring that thymus index, spleen index, activity of T lymphocyte, the level of IL-2 and IL-7 were distinctly higher, and the level of IL-10 were distinctly lower in MSC group, compared with aging model group (P<0.05). Compare with control group, the percentage of CD4+ CD8+ T cells reduced in model group; while the percentage increased in MSCs group, compared with model group(P<0.05). Detection of transmission electron microscopy showed:the thymus organization structure have been improved obviously in MSCs group, compared with model group (P<0.05)④Bone density scans showed that whole body and Femoral bone mineral density were higher in MSCs group, compared with model group (P＜0.05). Observation of trabeculae with scanning electron microscopy:in model group, the trabeculae of the femur became thinner, fragile, discontinuous with reduced quantity as compared with those in control group. The rats in treatment group had greater number of the trabeculae, resorption surface decreased and the collagenous fiber were much more regular than those in model group.PartⅣ:The mechanism of MSCs on aging ratsCompared with model group, the expression of P16,P21 decreased, and the expression of PCNA increased in MSCs group (P＜0.05). Western blot detected by the application of various organizations, the protein of P16,P21 decreased, and the protein of PCNA rised in MSCs group, compared with model group.Conclusion1. MSCs were onveniently and quiekly gotten by the way of bone marow adherent culture. The cultured cells were identified to be MSCs by morphology, the expression of cell surface marker, osteoblastic and adipogenic inducing. The bone marrow adherent culture method might be an ideal method for isolation and cultivation of MSCs in vitro.2. Mesenchymal stem cells expereience a gradual loss in its proliferation and replicate ability,which is related with senescence.3. The fluorescence of GFP is stable and remained strong express for 4 weeks, and the GFP has no negative effects on the MSCs cells. It is an ideal maker of stem cells and could be used for the study of MSCs transplantation. The expression of GFP was located in the various organs of aging rats, and persisted for a long time.4. Mesenchymal stem cells can repair tissue damage in aging rats caused by D-galactose, detected from function level, biochemical levels and cell level.5. MSCs may induce the expression of p16,P21 reduce, and the expression of PCNA increase. It maybe one of the mechanisms of MSCs anti-aging.The innovation of this study:1. MSCs could enhance immune function of aging rats; MSCs could improve osseous changes induced by D-galactose.2. Mesenchymal stem cells expereiences a gradual loss in its proliferation and replicate ability, which is related with senescence. So the therapeutic effect could be better to use yong rats' mesenchymal stem cells.3. MSCs may induce the expression of p16,P21 reduce, and the expression of PCNA increase, The mutual connection between these genes maybe constitute a cell ageing gene regulation chain. It maybe one of the mechanisms of MSCs anti-aging.