The Primary Study On Differentiation Of Bone Marrow Stromal Cell By Glucocorticoid

IntroductionThe application of glucocrticoid occupied the primary position among many causes of induced avascular osteonecrosis. Glucocorticoids have fine characteristics of anti-inflammation and immunosuppression, and have good action in therapy of rhemuatoid arthritis and spinal cord injury. The extensive application of glucocorticiods has resulted in the increasing incidence of non-traumatic avascular osteonecrosis. However, the precise pathobiological mechanism underlyig the induction of avascular osteonecrosis by steriods has not been eluciated to our knowledge. Various pathomechanism of avascular osteonecrosis by glucocorticoids include progressive accumulation of marrow fat stores, intraosseous hypertension, decreased infusion of blood supply, the increased amount of platelet and viscosity resulted in marrow vessel obliteration. These changes would make stem cells injuried, and then they could not differentiate into osteoblasts or chondrocytes, leading to the reduced resource of osteoblasts'precuors, or the dynamic imbalance of bone reconstruction and resorption, and with osteocytes apoptosis by steroid, further generating avascular osteonecrosis. In these theories, the research about accumulatoion of fat and increasing size of adipocyte in medullary cavity due to fafty metabolism or to the direct influence on differentiation of mesenchymal cell by steroid is becoming a main issue.Bone marrow mesenchymal cells(BMC) located in medullary cavity are the multipotentional cells, and able to differentiate into various tissuese and cells that derived from mesoderm or neuroectoderm, including osteoblast, chondrocyte, adipocyte and neurone. Cultured BMCs invitro have been verified that may be differentiate into osteoblasts and adipocytes in vivo and vitro, furthermore, the trend of differentiation would be changed with influence of microenviroment. The significant effect of glucocorticoid on the adipogenic and osteoblastic differentiation of BMCs is a hotspot recently. Core binding factor al (Runx2/Cbfal) is a key regulatory factor that is required to commit mesenchymal progenitors to the osteoblast lineage. By contrast, the expression of PPARy-2, the early specific adipocytic marker, destines cells for adipocyte differentiation. The expression and occurrence of the two genes could really govern and respond to the progress of mesenchymal cells. The dissertation observe and detect the effect of differentiation of bone marrow mesenchymal cell by glucocorticoid,to study and research the pathomechanism of non-traumatic avascular osteonecrosis with glucocorticoid. Furthermore, interpretation of the accumulation and increasing volume of fat is adipogenesis of mesenchymal cells by glucocorticoid.Study 1:The effect of dexamethasone on differentiation of bone marrow mesenchymal cellsBMCs have the abilities of multipotentional differentiation. Under different conditions, it can be induced to differentiate into various tissuese and cells. Most BMCs could differentiate into osteoblasts under normal culture conditions without dexamathasone in vitro. Therefore the experiment by dose and time-manners detect the effect of different concentration dexamethasone on differentiation of mesenchymal cells, and observe the microvariation of cells and supermicrovariation of cellular organs with a inverted phase contrast microscope and transmission electron microscope in the differentiation course.Study 2:Dexamethasone effects on the expression of osteogenetic and adipogenic genes of bone marrow mesenchymal cellsRunx2 gene, a key factor, regulates osteoblastic differentiation. PPARy-2 gene is fat-cell early specific marker. If expression of these genes has disparity, it will suggest diffferent trend of differentiation of mesenchymal cells. The experiment tests the variant expression of Runx2 and PPARy-2 genes under different concentration of dexamethasone stimulation using real-time PCR and Western blot methods, and compare with control group without dexamethasone, indicating the effect of glucocorticoid on cell differentiation at the levels of gene and protein.Study 3:Up-regulation of Runx2 gene supresses glucocorticoid-induced the adipogenic differentiation of mesenchymal cellGlucocorticoids enhanced adipogenic differentiation at the expense of osteoblastic differentiation in BMC, with increasing the expression of PPARγ-2 and decreased the expression Runx2 genes. How can we reverse the direction of adipogenic differentiation and find a solution for clinical administration? Therefore, we investigate expression of osteoblast marker genes(ALP, Col I, OCN) under Runx2 gene transfected condition, and explore influence of glucocorticoid on differentiation of mesenchymal cell transfected gene.Materials and MethodsStudy 1:1. Isolation and culture of bone marrow mesnechymal cellsThe bone marrow cells were flushed from the shat of bi-femur and tibia of 3-4w SD(SPF) with DMEM containing 10% fetal bovine serum, filtered, centrifuged and cultured. The stromal cells were cultured 7 to 10 days until they reached 80 to 90% confluence, and then digested with 0.25% trypsin and subcultured.2. Alkaline phosphatase and Sudan III counterstained:Divide four concentratiations of Dex duration to 21d and then Alkaline phosphatase, Sudan III counterstained, and to observe the number of adipocyte and osteoblast, compared with control without dexamethasone.3. Morphologic changes:Observe the microvariation of cell and supermicrovaria-tion of cellular organs under dexamethasone stimulation with a invert phase contrast and TEM microscopes.4. The activity of phosphatase assay:Use the improved Kaplow's method to test the activity of alkaline phosphatase of each group and then compare with control group without dexamethasone.5. MTT method determination of different concentrations of dexamethasone on the ability of cell proliferation.Study 2:1. We tested the expression of osteoblastic genes Runx2 and adipogenic gene PPARy-2 by using Real Time PCR, and then compare different concentration dexame-thasone groups with control.2. Western blot analysis was applied to investigate the differential expression of proteins (Runx2), and then at seven day after cultuerd with dexamethasone 10-7mol/L compare with control.Study 3:1. pCMV/flag-Runx2 plasmid reconstruction2. In order to confirm that Runx2 gene was transfected into BMC, we detect the expression of protein Flag, a plasmid tag, and protein Runx2 and osteocalcin by western blot and immunofluoresence techni, respectively.3. We test expression of osteoblast-related down-stream genes including ALP,ColⅠ,OCN and adipogenic genes of PPARy-2 and aP2 of cells transfected Runx2 gene by RT-PCR.4. By using western blot analysis, we test the expression of proteins OCN of BMC transfected Runx2 gene under stimulation of dexamethasone, to compare with control.Results1. BMCs exhibited fibroblast-like spindle phenotype without obvious deformation among subculture passages. However, when they were treated with dexamethasone, their shapes were became polygonal or irregular phenotype, and compacted alignment became agglomeration as well as increasing refractivity.2. With the increased concentration of Dex, the number of Sudan III stained lipochondria is significantly to rise.3. The activity of ALP without Dex is 1.57,4.49,5.0 times compared with 10-8, 10-7,10-6mol/l Dex groups.4. The expression of Runx2 gene decreased to 88-37% with 10-9M mol/1 to 10-6mol/l dexamethasone(P<0.05) and increased adipogenic genes 108-230% (P< 0.05) compared with control group without Dex.5. The result of TEM:appearance of nuclear pyknosis and membrance reductus, sparse cytoplasm, decreased the number of cellular organelles.6. Expression of Runx2 gene in BMCs with transfected technique has increased to 257%(P<0.05) compared with control, and adipogenic genes reached to 76% (P<0.05) and osteoblast-related down-stream genes distinctly increased (P<0.05).Conclusion 1.Dexamethasone enhance adipogenic differentiation of bone marrow mesenchymal cells.2. Demathasone affect the osteogenesis through mesnechymal cell gene experssion with decreasing the expression of Runx2 and increasing the expression of PPARy-2.3.Mesenchymal cell transfected Runx2 gene was resistant to adipogenic differentiation of BMC treated with high concentratrion Dex.