| BackgroundIn 2003, Prof. Yuan Lin has put forward a new hypothesis, fasciology. In fasciology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells of unspecialized connective tissues. The other 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. The supporting-storing system throughout the body regulates the functional and living status of differentiated cells and provides a stable internal environment for the survival of these cells. Adipose tissue is a an important part of the supporting-storing system, and adipose-derived mesenchymal stem cells (ADSCs) are main stem cells reserved in this system. We investigated the difference of stem cells reserved in different site, for example, in greater omentums and subcutaneous adipose tissues, by comparing the characteristics of multilineage differentiation potential and cell surface markers of ADSCs. This research will contribute to fasciaology and cell therapy.In recent years, interest has rapid grown in the research of ADSCs. Being one kind of mesenchymal stem cells, ADSCs have the capacity to self renew for indefinite periods and can differentiate to many different cell types. Mesenchymal stem cells can be isolated from several organs, such as bone marrow, fat, umbilical cord blood, peripheral blood and skeletal muscles. A significant number of investigations have focused on mesenchymal stem cells derived from bone marrow, which can differentiate to multiple cell phenotype, including bone, fat, cartilage, skeletal muscle and neural progenitor, under appropriate culturing conditions. Bone marrow mesenchymal stem cells were first identified and are one of the most widely used stem cell sources. Therapeutic potential of transplantation of them is invigorating. Compared with bone marrow mesenchymal stem cells, ADSCs do have an equal potential to differentiate into multiple cell phenotype. However, the simple surgical procedure, the easy and repeatable access to the subcutaneous adipose tissue, and the uncomplicated enzyme-based isolation procedures make ADSCs most attractive for researchers. Being a new source of therapeutic stem cells, ADSCs should be given more attention to.Autologous ADSCs as seed cells are ideal for autologous stem cell transplantation. In order to obtain a large number of autologous seeded cells, ADSCs should be idolated from adipose tissue and cultured in vitro. As the ADSCs donor, organisms are often in pathological situation (such as trauma). It should be investigate that whether there are sufficient ADSCs in such situation and the influence of trauma on the multiple differentiations and cell surface markers.Tissue and cell physiological behavior is influenced by the physical stimulation and chemical factors. The study shows that the suitable mechanical stimulation can cause bone quality and bone mineral density increases. And long-term weightlessness or bed criterion can cause bone quality loss. Moreover, the mechanics in different conditions, bone cell number and matrix synthesis ability changing, can cause the internal structure change of bone. Many vitro experiments already proved, the kinds, frequency, size and time, and many other factors of stress will influence the osteoblast biochemical response.Objective1. To isolate and cultivate ADSCs from SD rat and conduct immunophenotypic characterization with the following cell surface markers:CD11b, CD29, CD45, CD49d, CD90 and CD 106. And we use adipogenic and osteogenic induced differentiation of ADSCs to conform their differentiation potential.2. ADSCs of SD rats and Wistar rats from inguinal fat pads were isolated and cultured in vitro. In order to compare two commonly used ADSCs of differentiation of rat ability and get more evidences for the researches of ADSCs, the ADSCs of 2 kinds of rats were cultured under adipogenic and osteogenic condition to conform their differentiation potential.3. In order to explore the influence of ADSCs proliferation of tensile stimulation, and to discuss the dynamics change of osteogenic proliferation of ADSCs, we use low frequency tensile stimulate by Flexercell 5000 flexible basal loading system working on osteogenic ADSCs.Methods1. The isolation and culture of ADSCs of SD rats, multi potentiality and the characterization of cell surface markers:ADSCs of 6 SD rats from inguinal fat pads were isolated and cultured. Briefly, the adipose tissues were mechanically dissociated and digested with collagenaseâ… . The adipocytes were separated from the stromal vascular fraction by centrifuging the suspension. The cells in the stromal vascular fraction were plated in flasks at a density of 8000-10,000 cells/cm2. The cells were cultured with DMEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. After 24 hours, the non-adherent cells were removed and the adherent cells were expanded by serial passage. The morphological characterization of ADSCs was observed using phase-contrast microscopy.ADSCs at 4th generation were cultured under adipogenic and osteogenic condition to conform their differentiation potential. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The immunophenotypic marker including CD11b, CD29, CD45, CD49d, CD90 and CD106 of ADSCs grown for 4 passages was determined using flow cytometry. 2. We take inguinal fat pads from SD rats and Wistar rats in sterile conditions, and culture for ADSCs from adipose. The two kinds of ADSCs in 4th generation are taken to adipogenic and osteogenic induction. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation.3. We culture the 4th generation ADSCs in Bioflex six holes culture plates with osteogenic condition medium. The cells of stretching group and control group are tensile loading respectively every day. The cells of blank controls don't stretch stimulation and just use DMEM supplemented with 10% fetal calf serum. In 2d,4d, 8d,16d, after loading, we use the microplate method and AKP staining to detect the alkaline phosphatase (AKP) activity of ADSCs.Results1. Primary culture cells showed fibroblastic-like morphologic characteristics. From 1st to 4th generation, ADSCs appeared as a monolayer of large, flat cells, and became relatively homogeneous in appearance as the cells. ADSCs had the ability to differentiate along adipogenic and osteogenic lineages. When cultured under adipogenic condition for 2 weeks they were induced toward the adipogenic lineage. A fraction of the cells contained multiple, intracellular lipid-filled droplets that accumulated Oil Red-O. The Oil Red O-containing ADSCs exhibited an expanded morphology with the majority of the intracellular volume occupied by lipid droplets, consistent with the phenotype of mature adipocytes. When cultured ADSCs were exposed to an osteogenic induction medium, they aggregated and formed calcium deposits after 2 weeks. An alizarin red stain for precipitated calcium salt was performed on differentiated cells. Calcification appears as red regions within the cell monolayer. The immunophenotypic analysis revealed that the rat ADSCs were positive for CD29 and CD90, but negative for CD11b, CD45, CD49d and CD 106.2. Both of two kind rats can get ADSCs. No obvious difference between the original generation cells and 4th generation cells in microscope observation. Both ADSCs cultured under adipogenic and osteogenic induction medium for 2 weeks can be stained by Oil Red-O and alizarin red staining for precipitated calcium salt. 3. Compared stretching group with control group after 2 days, the AKP activity is higher. Along with the increase of cell numbers, AKP activity become corresponding strengthen. While cell numbers got to the peak, AKP activity became moderate. Through Alkaline Phosphatase dyeing method, the number of positive cells of stretching group is more than control group under tensile stimulation. But obviously with the extension of induction time, the positive cells in stretching group become close to the number of control group, but are higher than the negative control group.Conclusions1. The ADSCs harvested from SD rat obtained through an enzyme-based isolation procedures have the ability to differentiate along adipogenic and osteogenic lineages.. The results revealed the immunophenotypic characterization of them is consistent with mesenchymal stem cells.2. Both of two kind rats can get ADSCs. No obvious difference between the original generation cells and 4th generation cells in microscope observation. Two kinds of ADSCs both have the ability to differentiate along adipogenic and osteogenic lineages.3. Tensile stress stimulation can promote the activation, proliferation and alkaline phosphatase secretion of osteoblast differentiation of ADSCs in vitro. Tensile stress can promote osteoblast differentiation of ADSCs. Positive cells of alkaline phosphatase dyeing in stretching group are obviously more than controls. But with the extension of time, the number of positive cells in stretching group is gradually close to the number of positive cells in control group. But they are significantly higher than the blank control group. |
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