| Background:Aging is the decline of the whole body morphology and physiological function, which is a dynamic, complex process. Currently, prevention and anti-aging have become a focus of life sciences. Research on the mechanisms of aging have been the topics at the forefront of research in the field of biology, There are variety of theories explain the mechanism of geriatrics, such as the free radical theory, metabolic theory, immune system degradation doctrine, telomerase, and so on. But there is not a theory can complete explain the mechanism of aging.In fasciology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of is constituted of undifferentiated cells from the network of unspecialized connective tissues (fascia network). The other one is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. Undifferentiated stem cells in the supporting-storing system incessantly migrate to target areas, differentiate into committed-stem cells, and further differentiate into functional cells. The supporting-storing system provides energy and cell reserve for the functional system. The structures and functions of an organism are maintained by the incessant supplement and refreshment from the supporting-storing system to the functional system. Meanwhile, under the regulation of the nervous system and immune system, the fascia network throughout the body regulates the functional and living status of cells and provides a stable environment for the survival of functional cells. According to fasciology, the process of a life is just that of continuous consumption of the supporting-storing system. The supplementary of functional cells to repair the functions of damaged tissues ensures normal activities of the functional system. Therefore, how to maintain the normal state of fascia, continuously to provide a stable source of repair cells for the functional system and to maintain the normal function of cell division, maybe is the key to keep the human body with a longer life cycle. During the process of aging, if new cells are provided timely to the functional system, they will improve the quality and prolong the cycle of life. Unlike other theories of aging, the the fasciology from a different angle-Developmental Biology of Aging proposed a new understanding, with various theories of aging from the microscopic point of view, there is no contradiction that various microscopic point of the change. The final result is fascia tissue-support reserve system function decline.Mesenchymal stem cells have the capability of self-renewal and multi-directional differentiation. Initially, it was found by Friendenstein, is proved that in vitro they can differentiate into osteoblast and fat cells. And then the other research team found bone marrow-derived Mesenchymal stem cells (B-MSCs) can not only differentiate into tissues from mesoderm, but also into the tissues from both endoderm and ectoderm. It is consistent from Mesenchymal stem cells’multiple sources to the fascial connective tissue’s distribution, and from its biological characteristics of vertical and horizontal differentiation to the fascia connective tissue playing support and reserve role in body. ADSCs are main undifferentiated cells in the supporting-storing system. They are easy to be cultured, rich in source and low in immunity. They also have the capacity of cross-mesodermal differentiation and have endocrine functions. The potential of multi-differentiation of ADSCs makes them have developmental plasticity and makes them become a candidate of allograft. The adipose-derived stem cells can adjust the local microenvironment of the damaged tissue. Adipose-derived stem cells can secrete a series of cytokines and growth factor into the surrounding tissue fluid, to regulate tissue micro-environment, the formation of the micro-environment that is conducive to the growth of stem cells-stem cell niche, recruitment of endogenous stem cells from receptor to reach the target and to differentiated tissue cells directed to the specific needs. Adipose stem cells themselves can withstand hypoxia, can release the same time in a hypoxic environment antioxidants, free radical scavengers, molecular chaperones, heat shock proteins, etc., removal of damaged local cytotoxic substance to promote still alive cells recovery form conducive to fat grafting tissue cells to survive and function to maintain the microenvironment.ADSC may also paracrine cytokines promote tissue repair. Intravenous injection of ADSC autonomously gathered at the lesion site involved in the repair of damage. ADSC This "homing" role, the exact mechanism is unknown. Reported that by increasing the expression of membrane-type matrix metalloproteinase1(membranetype1matrix metallop roteinase, MT1-MMP) and ADSCs promote activation of MMP-2zymogen enhances migration. Recent studies found that, ADSC release of new mitochondria to damage cells, thereby saving the process of aerobic metabolism. This shows the the ADSC is available through a variety of mechanisms involved in tissue repair. In a word, ADSCs secrete various growth factors. These proteins control and manage the damaged neighboring cells. Meanwhile, the production and secretion of growth factors has been considered as essential function of ADSCs. ADSCs also exhibit an antioxidant effect, although the mechanism is not yet clear. When ADSCs are tansplanted to the damaged animals, these stem cells may secret cytokines and growth factors to stimulate the animal to repair the damaged tissues. ADSCs have been found to produce anti-oxidants, free radical scavenger, such as heat shock protein, to promote the repair of the survival cells. ADSCs can stimulate endogenous stem cells to supplement to the target organ or can direct migrate of their own to the repair region, by which to regulate microenvironments of stem cell and promote them differentiate into functional cells, so as to replace aged functional cells to maintain the balance of the body. Further studies are necessary to understand the complete protection mechanism of ADSCs.D-gal has been found to induce aging rats. Oversupply of D-galactose will result in abnormality of metabolism. The oxidative metabolism of D-galactose produces reactive oxygen species, which suppress the ability of the cells to eliminate them, consequently causing impairment of cellular membrane, structure, and gene expression. D-gal also can impact on cell metabolism and function of some important enzymes, which contributes to making clear the mechanism of this aging model to some extent. Its specific mechanisms of aging and functional decline may be related to metabolic disorders, immunological damage, free radical damage and mitochondrial injury related.Disorders of glucose metabolism will cause abnormal heart, liver, kidney, brain and other important organs metabolism, finally appeared senescence. D-galactose model because of the full impact of cell metabolism and some important enzyme function, caused by the aging more comprehensive. The specific mechanisms of aging and functional decline may be related to metabolic disorders, immune injury, free radical damage and mitochondrial injury related. At present, the model has been widely used in research on the mechanism of aging and delaying senility drug screening. D-gal induced subacute aging model can simulate the brain, liver, kidney, skin, gonadal axis, osteoporosis, the immune system and multiple organ damage. Reports in the literature can be caused by a variety of methods extract of Chinese medicine, Chinese medicine decoction, hormones, and acupuncture treatment of this model of aging, also reported application of stem cell transplantation in the treatment of.Androgen can improve the excitability of the central nervous system, cause sexual impulse, thus maintaining the normal sexual function. Leydig cells (Leydig cells) is the main cell body of testosterone secretion, secretory volume accounted for about95%of total body testosterone; treatment of reproductive disorders of the male international will also focus on cultivating Leydig cells and transplantation. Leydig cell has the characteristics of synthesis, secretion of testosterone, testosterone synthesis process,-HSD317β,3β-HSD1catalytic DHEA formation of testosterone. And3β-HSD is a key enzyme in the synthesis of testosterone in adult rat testis, only mesenchymal cells expression of3β-HSD, so the current identification of mesenchymal cells generally use3β-HSD specific staining method. Leydig cell separation Percoll gradient concentrations of large, high purity, good cell vitality, in solving the problem of androgen secretion cell source for tissue seed to construct tissue engineering technology. Tissue engineering of androgen is obtained with the method of Leydig cell and the construction of the secretory tissue can maintain testosterone secretion in longer period.The aim of this experiment is to investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms, and to verify the theory of the fasciology.In order to study the exogenous adipose-derived stem cell transplantation is a function of delaying senility and its mechanism of action, this experiment in vivo transplantation of exogenous ADSCs on D-galactose (D-gal) effects of aging rats free radicals and immune senescence index and the ability of aging rats improvement; adipose-derived stem cells in vitro matrix D-gal treatment on Leydig cells (LCs) after co-culture, exploring the protective effects of ADSCs on Leydig cell, to explore a new anti-aging mechanism, to provide new ideas and methods for clinical aging treatment.ObjectiveTo investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms and to verify the theory of the fasciaology.1. To judge that whether the ADSCs exist in facial connective tissue;2. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on free radical metabolism and immune function of rat aging model induced by D-gal from fasiaology perspective; to explore a new method for anti-aging.3. Through mating experiments, to observe if ADSCs from fascia connective tissue can improve the aging rats induced by D-galactose sexual function; by immunohistochemical staining to location the transplanted ADSCs, labeled stem cells in the testis and displacement, and the secretion of Leydig cells of3β-HSD, 17beta-HSD. By immunohistochemical staining, morphological observation of cell changes of Leydig cells. Observation of testosterone secretion; Using Elisa to test the secretion of3β-HSD andl7p-HSD.4. Isolation and culture of Leydig cells in vitro, to observe the protective effect of adipose-derived stem cells on D-gal treated Leydig cells.5. Rut female rats evoke male rats to mate, to observe male rats’mate ability and mate number of times. Through radioimmunochemistry to detect testosterone content of the rats’blood serum. Observation of the expressionã€location and translocation of ADSCs in the testis by immunohistochemistry staining, And provide the evidence for the theory of "fasciology".Methods1.To detach the ADSCs from the rats’adipose tissue, to cultivate the cells in vitro and observe their formation, means of growth and biological markers on the cells’membrane, and to induce ADSCs to differentiate into adipogenic and osteogenesis, and observe their formation and specific markers. To judge whether connective tissue contains ADSCs?2. In vitro Percoll separating medium gradient isolation and culture of Leydig cells, and identified by morphological and3β-HSD specific staining;0,4,8,12,16g/L of different concentrations of D-gal treating Leydig cells for24h, the cell survival rate was calculated, screening the best concentration simulation cell aging model; flow cytometry detection the the changes of cell cycle of8g/L D-galactose treatment Leydig cell in0,24,48,72h different time points.3. Isolated and cultured in vitro, the4th generation ADSCs, then preparation adipose-derived stem cell matrix (ADSC-CM); while using8g/L D-galactose and adipose stem cells matrix (ADSC-CM) co-culture Leydig cells for24h, to detect the cell activity by MTT; cells were co-culture for24h, then detected by flow cytometry; co-culture of48h detection of cell senescence using SA-P-gal anhydride enzyme staining; immunohistochemical study of3β-HSD1,17β-HSD3secretion; secretion of testosterone。 4. Thirty male SD rats weighing180-200gwere randomly divided into3groups (A, B and C),30in each group. D-gal was dissolved in0.9%saline at a concentration of15%just before use.The rats in groups B and C were subcutaneously injected with D-gal at a dose of1000mg/kg body weight once every day for a total of8weeks. The rats in group A were injected with physiological saline at the same volume everyday for8weeks for experimental control.5. Harvest and culture purified ADSCs from SD rats and these cells were labeled with Brdu for use. After all D-gal injections have been finished, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the4th passage were harvested and resuspended in the physiological saline at3×106cells/ml. Each rat in group C was injected with3x106of ADSCs into its tail vein. The rats in groups A and B were similarly administered with same volume of physiological saline.6. Female rats were brought into behavioral estrus with sc injections of200μg/kg estradiol benzoate48h before and2mg/kg progesterone4h before testing. Only fully receptive females were used to copulate with males. The changes of structure of the testis were showed by HE stain. The changes of30-HSD expression and apototic index of leydig cells were analyzed by immunohistochemistry. The transplanted ADSCs were detect by Brdu immunohistochemistry.7. Statistical analysis was carried out using SPSS13.0statistical software, ANOVA followed by Fisher’s post hoc analysis. variance, the mean were compared with single factor analysis of variance, two-two compared with LSD method; variance not neat, multi-average number were compared by Welch method, two-two were compared with Dunnett’s T3, P<0.05were significant differences.Results1. ADSCs can be extracted from the rats’fat tissue. In primary culture, the isolated ADSCs exhibited a fibroblast-like morphology.The two passage cells were adherent and had a spindle-shaped morphology in the third days; Flow cytometric analysis demonstrated that ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens; Culturing of undifferentiated cells for14days under adipogenic conditions induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were characteristics of adipocytes. Induction of osteogenic differentiation of the cells for14days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were characteristics of osteoblasts.2. In vitro experiment, Leydig cells can be successfully cultured by Perpoll separation method. Leydig cells were polygon, triangle. Monolayer growth, stretching in whorls, cells in the colony form proliferation; By3β-HSD specific staining, Leydig cells are stained blue-black under microscope, and other testicular cells do not stain, Leydig cell purity can reach more than95%; MTT showed8g/L D-gal cell survival rate is the most suitable simulation model can successfully induce cell senescence, induce Leydig cells aging; with the extension of time,8g/L D-gal LCs of G0/G1phase cells were increased, S phase cells decreased.3. ADSCs-CM was extracted with ADSCs supernatant; Leydig cells after8g/L D-gal co-culture, MTT showed improved cell activity; when co-cultured for24h, flow cytometry showed the percentage of the G0/G1phase cell decline in the cell cycle; co-culture of48h, SA-β-gal anhydride enzyme staining positive cells, co-culture galactose anhydride enzyme group SA-beta reduction, cell senescence reduced; immunohistochemistry examination showed cells secrete3β-HSD1,17β-HSD3increase.4. In this experiment, aging model has been successfully established by chronically exposing animals to D-gal. After transplanting ADSCs, the rats in cell treatment group also showed a significant decrease in MDA activities and a significant increase in SOD levels and IL-2levels. Thymus index and spleen index also have been decreased. While the NO content have no change by contrast with the aging group.5. We found Brdu positive cells distributed in testis tissue of experimental group SD rats after transplantion and trends to migrate to the injury area. The mate ability of aging rats have been improved by transplanting ADSCs. HE staining showed seminiferous tubules in aging group were thinner than that in normal control groups and the sperm number in the tubules decreased apparently. Immunohistochemistry showed that the level of3β-HSD expression reduced significantly compared with that in the control groups. TUNEL assay showed that ADSCs reduced apoptotic leydig cell death induced by D-gal. The testosterone contents and3β-HSD expression in cell treatment group have been significantly increased by contras with the aging group.Conclusion1. Fascia connective tissue derived mesenchymal stem cells have the morphological features of long spindle, and polygon, and can form cell colony. There is no significant difference with the other kinds of MSCs in morphology; ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens, in line with mesenchymal stem cell characteristics; the cells can be induced to differentiate into osteoblasts and fat cells. It can be judged that mesenchymal stem cells exist in mature rats’facial connective tissue.2. Through the method of Percoll gradient centrifugation, can obtain the high purity of Leydig cells; Leydig cells can be identified by3β-HSD specific staining,.3. The concentration of D-gal can ensure the survival rate of cells, induce cell senescence. Under high concentration environment (16g/L) and concentration (8g/L) of the long time effect, can induce apoptosis in Leydig cells.4. ADSCs-CM and8g/L D-gal treatment of Leydig cells, ADSCs-CM can effectively protect Leydig cell from D-galactose damage; ADSCs can increase cell activity, reduce the number of cell senescence. Promote the secretion of3β-HSD,17P-HSD, protect Leydig cells.5. Rodents injected with D-gal for6-10weeks can successfully establish Sub-acute aging model, which shows progressive deterioration of learning and memory capacity and mate ability, increased production of free radicals in the body.6. Through transplanting ADSCs to the aging rats, SOD levelsã€IL-2levels〠thymus index and spleen index have a significant increase in cell treatment group rats while contrast with the aging model rats. While MDA activities show a significant decreased. 7. Transplanting ADSCs can improve the mate ability of aging rats, stimulate the increase of the testosterone contents and3β-HSD expression, improve the quality of life.In summary, the research initiated to study the effects of transplanting ADSCs to D-gal induced aging rats on free radical metabolismã€immune functionã€the capability of spatial learning and memory and mate ability of rats aging model induced by D-gal from fasiology perspective, through cell culture, HE staining, immunhistochemistry technology.That to explore the mechanism of transplanting ADSCs to aging rats is a new try of anti-aging therapies in the point of fasiology. In this experiment,supplementing the D-gal induced aging rats with exogenous stem cells can delay the aging procedure, improve the quality of the life. |
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