The Effects Of Transplanting Adipose-derived Stem Cells To D-galactose Induced Aging Rats

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. In fasciaology, 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.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. 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.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.To 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.To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on the aging rats as below:(i) whether they improved the behavior of aging rats in MWM test; (ii) whether they decreased neronal degeneration and lowered AI in the rat brains.4. 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 expression location and translocation of ADSCs in the testis by immunohistochemistry staining. And provide the evidence for the theory of "fasciaology".1. 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. Ninety male SD rats weighing 180-200 g were randomly divided into 3 groups (A, B and C),30 in each group. D-gal was dissolved in 0.9% saline at a concentration of 15% just before use. The rats in groups B and C were subcutaneously injected with D-gal at a dose of 1000 mg/kg body weight once every day for a total of 8 weeks. The rats in group A were injected with physiological saline at the same volume everyday for 8 weeks for experimental control.3. 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 the 4th passage were harvested and resuspended in the physiological saline at 3×106cells/ml. Each rat in group C was injected with 3×106 of ADSCs into its tail vein. The rats in groups A and B were similarly administered with same volume of physiological saline.4. MWM was used for behavioral testing of rats, which was subsequently conducted between 9:00 and 17:00 for 5 days. The paraffin sections of brains were prepared for hematoxylin and eosin (H&E) staining and for terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method. The apoptotic indexes (AI) was also calculated for statistics. Then to explore whether transplanting ADSCs can decrease neronal degeneration and lower AI in the rat brains.5. Female rats were brought into behavioral estrus with sc injections of 200μg /kg estradiol benzoate 48h before and 2mg/kg progesterone 4h 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 of 3P-HSD expression and apototic index of leydig cells were analyzed by immunohistochemistry. The transplanted ADSCs were detect by Brdu immunohistochemistry1. 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 CD 106, and negative for CD 49d, CD11b and CD45 surface antigens; Culturing of undifferentiated cells for 14 days 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 for 14 days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were characteristics of osteoblasts.2. 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-2 levels. Thymus index and spleen index also have been decreased. While the NO content have no change by contrast with the aging group.3. The rats in aging group showed a poor behavior in Morris water maze, which suggests that these rats suffer problems in spatial learning and memory. While cell treatment group have better performances. TUNEL assay showed that ADSCs reduced apoptotic neuronal cell death induced by D-gal. This suggests that the transplanted ADSCs have an important protective effect against D-gal-induced apoptosis in pyramidal and granular cells.4. 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 of 3β-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 and 3β-HSD expression in cell treatment group have been significantly increased by contras with the aging group.1. 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 CD 106, and negative for CD 49d, CDllb and CD45 surface 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. Rodents injected with D-gal for 6-10 weeks 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.3. Through transplanting ADSCs to the aging rats, SOD levels,IL-2 levels,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.4. In Morris water maze test, the ability of spatial learning and memory in cell treatment group has been improved. And Tunel showed reduced apoptotic neuronal cell death, which suggests that the transplanted ADSCs have an important protective effect against D-gal-induced apoptosis in pyramidal and granular cells.5. Transplanting ADSCs can improve the mate ability of aging rats, stimulate the increase of the testosterone contents and 3P-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 metabolism,immune function,the capability of spatial learning and memory and mate ability of rats aging model induced by D-gal from fasiaology 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 fasiaology. 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.