Treatment Of Osteoporosis And Screening Of Differential Protein In Osteoblast Differentiation Of Adipose-derived Stem Cells

BackgroundOsteoporosis is a metabolic disease characterized by increased osteopsathyrosis and easy to fracture caused by osteopenia and destroy of bone micro-architecture. As the population get ageing, the osteoporosis patients increase. It is of great clinical value to research the prevention and treatment of osteoporosis.Professor Yuan Lin put forward the theory of fasciology, which holds that the human body is composed of supporting-storing system and functional system, the former of which is consisted of the undifferentiated non-specific fascia connective tissue and the latter of which is consisted of various functional cells. The functional system is based on the cells with specific function while the supporting-storing system is centered on the undifferentiated stem cells in the fascia network. The undifferentiated stem cells in supporting-storing system can differentiate into different directional stem cells, supplying as continuous cell source and nutrients for the functional system, maintaining the stability of the internal environment. Therefore, only supported by the supporting-storing system, can the structure and function be normally held. Once one kind of functional cells cannot be supplemented timely, the human body would suffer corresponding disease. In fasciology, osteoporosis is caused by insufficient supplement of bone formation from undifferentiated stem cells in fascia network after absorption. Therefore, it is crucial for osteoporosis treatment that how to increase the undifferentiated stem cells in fascia network and accelerate the directional differentiation of bone in the negative balance of bone metabolism, and to promote the bone formation to build the positive balance.Adipose tissue is an important reserve in the supporting-storing system, among which adipose-derived stem cells (ADSCs) are an important cell reserve, and it becomes a hotspot in stem cell research. It is demonstrated that ADSCs are easy to isolate and culture, and have various differentiation potential, even transdifferentiate into bone, hepatocyte, cardiocyte, cartilage, adipose, muscle and nerve. ADSCs have low immunogenicity and have the function of immunoregulation. In addition, compared with bone marrow-derived stem cells, ADSCs have the advantages of broader source and less injury and pain. These features make it possible for heteroplastic transplantation. Increasing the undifferentiated ADSCs and promoting them to differentiate into bone, accelerating the bone formation are possible ways of preventing and treating osteoporosis.Establishing the osteoporosis model by glucocorticoids has become mature. Long-term high doses of glucocorticoids can cause calcium and phosphorus metabolism disorders, reduction in osteoprotegerin secretion, increased secretion of parathyroid hormone, inhibition of the proliferation and activity of osteoblasts and increased quantity and activity of osteoclasts. For these reasons, the bone absorption increases and bone formation decreases, resulting in osteoporosis.The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. Proteomics can analyze the dynamic change in the cells and the composition of protein, the expression level and modification. It also can help with the research of interaction and correlation of different proteins, revealing the function of proteins and cell mechanics. As the molecular weight and net charge are irrelevant, dimensional electrophoresis can isolate the total protein in light of the two distinct differences. It can be used for detecting the expression change of protein rapidly and correctly.In order to research the prevention and treatment of osteoporosis by allogeneic ADSCs, as well as the protein mass spectra change in the osteogenesis induction from ADSCs, this experiment observed the effects of transplanting allogeneic ADSCs on glucocorticoids caused osteoporosis in the aspects of bone morphometry and protein mass spectra change. This experiment aimed to exploring a new research direction of prevention and treatment of osteoporosis.Objective1. To detach and culture the ADSCs from the adipose tissue of rats, identify of the multipotency of directional differentiation and surface markers to estimate the undifferentiated ADSCs in the supporting-storing system.2. To detect bone mineral density to estimate the success of osteoporosis model.3. To observe the effect of allogeneic ADSCs on glucocorticoid induced osteoporosis in rat bone tissue microenvironment by bone morphometry.4. Protein mass spectrum analysis on osteogenesis induction of ADSCs for2and3weeks.5. To provide experimental evidence for fasciology, which holds that mobilizing the undifferentiated stem cells in supporting-storing system could treat degenerative and senile diseases.Method1. To detach the ADSCs from adipose tissue of adult rats, isolate ADSCs in fascia by collagenase digestion and then cultured the cells in vitro. The6th passage cells were observed their morphology, differentiatial potent by induction of adipogenesis and osteogenesis, and their biological markers on the cell membrane, such as CD49d, CD90, CD11b, CD29, CD45and CD106.2.40female SD rats were randomly divided into4groups (A, B, C and D),10in each group. Rats in B, C and D groups were injected with prednisolone into the neck subcutaneously at a dose of8mg/kg of body weight, three times per week for12weeks. The rats in group A were injected with physiological saline at the same volume for12weeks. Group A and B were sacrificed and specimens were procured to judge whether model succeeded after12weeks.3. As the model was succesfully built, collect the ADSCs of6th passage and inject3×106ADSCs through the caudal vein of rats in group C, inject1ml saline into rats in group D. The rats were sacrificed4weeks later.4. Detach the upper1/3of right tibia, sagittally cut open, fix and undergo gradient dehydration. Then soak in immersion Ⅰ (methyl methacrylate70ml+dibutyl phthalate30ml), Ⅱ (immersion Ⅰ+benzoyl peroxide1g) and Ⅲ (immersion Ⅰ+benzoyl peroxide2.5g) for2days respectively and embed in fresh inmrnersion Ⅲ. Contineously slice into6μm thick with Jung K machine and observe histology and osteoid with Gemisa staining and von Kossa staining. The measurement area was1-4mm below the grouth plate. Bone histomorphometry is analyzed by LeciaQWin software. The parameters are ratio of BV/TV, Tb.Th, Tb.N, Tb.Sp and OC.N, which are calculated by internationally accepted formula.5. Separate the protein samples of non induced,2weeks,3weeks after induction with two-dimensional gel electrophoresis, silver stain and obtain the proteomic map of all kinds of cells. By comparing the three maps, the differential expression of protein can be found to undergo matrix-assisted laser desorption ionization time of flight, mass spectrometry peptide mass fingerprinting analysis and protein database information retrieval.Results1. Observed by optical microscope, most of the ADSCs of primary culture can adherent after24hours. The cells are round, polygon or fusiform. If cells grow well, they can be transferred at ratio1:3after7days’culture, and be transferred every3days afterwards. In6th passage, the ADSCs have the homogenicity with a spindle or polygon shaped. The cells can be induced to differentiate into fat cells and osteoblasts. Flow cytometry demonstrated that CD29and CD90were high positive, CD106weak positive, while CD11b、CD45and CD49d were negative.2. After administration of long-term and high-dose glucocorticoid, the bone trabecular of rats in model group was thinner and tangled, and destroyed with a wide intertrabecular distance, even had lacunae on the surface. The quantity, volume and thickness of bone trabecula were all decreased significantly, along with a significant increasing of trabecular separation and osteoclast number (P<0.05).3. Transplanted allograft ADSCs could significantly increase the bone trabecula number. The bone trabecula became thicker and harder. The quantity, volume and thickness of bone trabecula were all increased significantly but trabecula separation and osteoclast number decreased (P<0.05).4. Compared with undifferentiated group, there were54spots with same and more than twice variation trend after2and3weeks’ osteogenesis induction. There were12proteins up-regulated and15down-regulated in osteogenesis group.Conclusion1. There are ADSCs in the supporting-storing system, which are undifferentiated mesenchymal stem cells.2. Osteoporosis model can be built by long-term high-dose administration of glucocorticoid.3. Differential protein can be screened by osteogenesis induction of ADSCs.4. This experiment provides part experimental evidences for the fasciology hypothesis that mobilizing the undifferentiated stem cells in fascia supporting-storing system could treat regenerative diseases.To be concluded, transplanting ADSCs to glucocorticoid-induced osteoporosis rats have therapeutic effects on bone formation, increasing bone density, improving biomechanics property, enhancing bone strength, thereby decreasing incidence of bone fracture. The study further implicates that increasing quantity of stem cells and inducing directional differentiation of osteogenesis may be a new way of preventing and treating osteoporosis in clinic.