| Purpose:Steroid-induced avascular necrosis of bone marrow may be related to hormonal induction of mesenchymal stem cells (BMSCs) of adipogenic differentiation, chinese Herbal Medicine for the prevention and treatment of early disease have significant effect.Observe the effects of blood circulation through the active ingredients of saffron HSYA A (HSYA) of femoral head avascular necrosis associated indexes,research HSYA on steroid prevention and treatment of avascular necrosis of the role, chinese Herbal Medicine to further verify this femoral head necrosis of control effect, then explore the pathogenesis and mechanism of traditional Chinese medicine, Provide more effective clinical therapies and prescriptions of the experimental evidence.Method:12 healthy 30 dayses old SD big rats,Organization within the medullary cavity of femur whichever to Cultured bone marrow mesenchymal stem cells, Passage,purification,identification,the MTT method to get the HSYA of bone marrow mesenchymal stem cells the optimal concentration,with the best concentration of drug in the cell culture dose group,2 times the optimal concentration of HSYA measured as the high dose group,and 1/2 dose groups in the best concentration.The control was to DMEM containing 15% FBS culture medium;Control group containing 15% FBS DMEM,99ml+ dexamethasone stock solution lml culture medium intervention; High-dose group containing 15%FBS DMEM,99ml+ dexamethasone stock solution lml +0.2 mgHSYA medium intervention;Middle dose group containing 15% FBS DMEM,99ml+ dexamethasone stock solution 1ml +0.1 mgHSYA medium intervention;The low-dose group containing 15% FBS DMEM,99ml+dexamethasone stock solution 1ml+0.05 mgHSYA medium intervention.The 5 groups of above for 6 days for Line transmission electron microscopy, oil red O staining and triglyceride test, PCR assay of expression of PPARymRNA and aP2mRNA detection.Result:①Cell identification:Morphology light microscope observation of rat BMSCs, Cells showing a fibrous or spindle-shaped into,Adherent growth, cell growth are closely attached,Gradual integration into the film, ordered along the long axis of the cell body,was swirling.Purified by passage BMSC,BMSCs form of three generations of a single uniform, was typical of the polar fusion,whirlpool growth;By flow cytometry for CD45 and CD90 antigen positive sign expression detection test results showed:The adherent cells grew well in the first three generations in the surface markers of the line.3rd generation cell CD45 positive rate of about 2.0%, CD90 positive rate was 99.6%.It Can explain the present study, bone marrow adherent cells were isolated and cultured in vitro for the BMSCs.②Oil red O staining showed:The no control group was stained fat cells;The control group, fat cells staining most, Showed a cluster distribution.High, medium and low-dose group were found in varying amounts of staining of fat cells,Are all less than the control group.③Electron microscopy observed:Control group, high, medium and low dose group did not find fat cells,and there is no fat particlesin cells,Cell morphology was normal,The control group,see a lot of fat particles of intracellular accumulation.④Triglyceride test:control group,the triglyceride content of cells was significantly higher than the control group,The HSYA each group were significantly decreased compared with the control group,medication was no significant difference among the three groups.Compared with the control group,HSYA low, medium and high dose group was significantly lower than the control group.⑤Quantitative PCR:the PPARymRNA and aP2mRNA expression. one control group was significantly higher than PPARymRNA group, HSYA high and low dose group and control group there was a significant difference (P<0.01),compared with the control group,HSYA high dose group compared with the control group P> 0.05,no significant difference, the low-dose group compared with the control group P<0.01,there was significant difference.Ap2mRNA control group was significantly higher with the control group (P<0.01),The HSYA groups were high compared with control group P<0.05,there are significantly different;High, dose group compared with the control group P<0.05,significant differences.Low-dose group compared with the control group P> 0.05,so low-dose group and control group no significant difference.Conclusion:1.In this study, the cells were isolated and cultured in vitro morphology, flow cytometry and other surface markers are identified on BMSCs,And of high purity.2.The big amount of hormone inducement can urge BMSCs to become a fat to divide, make fat cell increase.3. HYS As interferes,Intracellular triglyceride content, PPARymRNA and aP2 mRNA expression were significantly lower than the control group,Oil red O staining, electron microscopy, see more fat cells and particles was seen after a simple intervention hormone fat cells and particles less,tip HSYA BMSCs inhibit hormone induced by the adipogenic differentiation.4. HSYA hormonal control mechanism of avascular necrosis of femoral head is not only to improve the microcirculation,BMSCs also inhibit adipogenic differentiation,to promote their osteogenic differentiation. |
