The Initial Analysis Of Differential Proteomic In The Osteoblast-like Cells Which Are Indued By Glucocorticoid Comparing With The Ones Treated By The Method Of Kindey-tonfying And Related Study

Research Objective:The necrosis of femoral head induced by steroid is known to all. With the steroid being widely used, the morbidity has been yearly increased. SANFH becomes the prodomainant disease in the nontraumatic necrosis of femoral head.Therefore, it is essential to research its pathogenesis and its mechanism of therapeutic effect.The exact pathogenesis of SANFH remains unclear.The following points of view is commonly accepted: the theory of fat embolism; the theory of the fatty degeneration of osteocyte; the theory of high pressrue and venous stasis inside the bone. All these theories are not enough to explain its mechanism. The research about it at the leve of protein molecular is not reported at present.The proteomics is a new and developing subject in the postgenometic era. Differential proteomics mainly aims to find the proteomic difference in the sample of cells or tissues at certain circumstance, detect the change in the process of the physiology or pathology, explore the machnism of the organize reflexing to the surrounding and cytoregulation,and acertain the nature or analyse the function of some symbolic protein. Once the sufficient information acquired, the reason why the change happens can be deduced in theory.The study tries to detect the differential proteomics which is affected by the steroid in osteoblast, to illustrate the pathogeny.This is a breakthrough in theory,which can provide theoritic guidance to the treatment of SANFH.As the glucocorticoid being used for long term or in overdose, the blood suffering heat is sticking and stagnant, the circulation is not smooth. The arteries and veins are injured, therefore, the blood is overflow from the vessel and stay in the femoral head, in which, the hematoma and arthromyodynia emerge. At meantime, the phlegmonosis stays in the kindey, and quell the kidney-essence.For above reasons, avascular necrosis of the femoral head developed. Most scholars regard SANFH being mainly due to renal deficiency or blood stasis. According to the theory of TCM which the kidney is in charge of bone,we apply the Rehmanniae pill of Six Ingredients as the formula invigorating the kidney,to affect the osteoblast-like cells.With the method of differential proteomics,it is illustrated the mechanism of SANFH happened and treated by the tonying kidney method.Research Method:1 Experimental study1.1 Effect of pastille serum of Rehmanniae pill of Six Ingredients on OS-732 cell proliferation and differentiation.Objective: To investigate the effect of kidney-tonifying herbs on bone osteoblast-like cells proliferation and differentiation. Methods: Preparing pastille serum of Rehmanniae pill of Six Ingredients with low, medium and high doses and using 10% pastille serum with different doses to cultivate OS-732 cells. Observe the cell growth using microscope; count cells and draw grouth curve; Dectect cell proliferation with MTT method; Determin the activity of cell alkaline phosphatase (ALP). Results: Cell growth of the pastille serum group is satisfactory, medium dose group has significant discrepancy compared with control group(P＜0.05); About cell proliferation and cell ALP activity determination, the medium dose group has significant discrepancy compared with control group(P＜0.05). Conclusion: Suitable dose of kidney-tonifying herbs can promote osteoblast proliferation and differentiation.1.2 Effect of different densities DMX on OS-732 cell proliferation and differentiationObjective: To investigate the effect of glucocorticoid on osteoblast-like cells proliferation and differentiation through DMX culture in vitro with different densities. Methods: Adding low, medium and high doses of DMX to cultivate OS-732 cells, using FCM to detect the cell apoptosis and analyze the cell cycle within 24h and 48h. Results: the low concentration DMX can cause the G₂+M period increase significantly compared with control group (P＜0.05) within 24h; the medium concentration DMX has no significant discrepancy compared with control group in all periods; The high concentration DMX has blocking effect on G0/G1 period and has significant discrepancy compared with control group(P＜0.05). Both the control group and DMX group showed apoptosis peaks, the cell apoptosis rate of the medium concentration group has no significant discrepancy compared with control group; the cell apoptosis rate of the low concentration group was significantly lower than control group (P＜0.05), the cell apoptosis rate of the high concentration group was significantly higher than control group (P＜0.05).The stuation within 48h is simular to that within 24h. Conclusion: Time and quantity of glucocorticoid used has different effects on osteoblast-like cells. Low dose of glucocorticoid (physiological need dose) has promoting effect on osteoblast-like proliferation and high dose (exceed the physiological need dose) has inhibitory effect on osteoblast-like cells proliferation and differentiation.1.3 Intervention effect observation on osteoblast-like cells with electron microscope kidney-tonifying methodObjective: To observe morphological changes of osteoblast-like cells and to understand histologyical difference by making comparison between control group with steriod group and herb group. Methods: Cultivating OS-732 cell in control groups steriod group and herb group, collecting and observing cell morphological changes by electron microscope. Results: irregular shape, less mitochondria and more vacuolus were observed in steriod group; cell morphology improved obviously with more mitochondria and endoplasmic reticulum in herb group. Conclusion: glucocorticoid can change the norphology of the osteoblast-like and affect the bone formation function of the cells. Kidney-tonifying herbs can improve the morphology of the osteoblast-like cells and reverse the apoptosis of the osteoblast-like cells.1.4 Protein initial analysis of hormone intervening effect difference on osteoblast-like cells using kidney-tonifying method.Objective: To investigate the protein spots discrepancy on two-dimensional electrophoretogram of the steriod group and herb group with control group and analyze the protein expression change of osteoblast-like cells apoptosis caused by steriod and effect by using herbs. Methods: Extracting the total protein of the OS-732 cells cultivated in control group, steriod group and herb group, analyzing the discrepant protein spots after two-dimensional electrophoresis. Results: There were 8 protein expression decreased in hormone group, spots of SSP1005, SSP2301, SSP3202 and SSP6501 decreased mostly. There was no protein expression in spot of SSP564; Protein expressing in the above-mentioned 7 spots were reinforced in different levels in herb group and spot SSP5604 was reinforced mostly. Conclusion: Effect of glucocorticoid can attenuate some certain protein expression of the osteoblast-like cells and cause apoptosis; Kidney-tonifying herbs can reinforce some protein expression.Kidney-tonifying herbs can promote osteoblast-like cells proliferation and differentiation, improve the morphology and structure of the osteoblast-like cells; Restrain osteoblast-like cells apoptosis induced by glucocorticoid and reinforce some certain protein expression caused by osteoblast-like cells apoptosis induced by glucocorticoid.2 Clinical studyObjective: to observethe therapeutic effect of SANFH treated by the tonifying kidney herb.Method: 66 patients divide into 2 groups. the control group is given shenmaichengu tablets, while the combine medication group is given Shenmaichengu tablets and Rehmanniae pill of Six Ingredients. Make the evaluation in the pain, the fuction, the joint range of motion and the X-ray comparing the two groups. Result: The evaluation in relieveing pain, improving fuction, adding the joint range of motion in the combine medication group is proir to that of the control group.However, there is no difference in the evaluation of X-ray.3 Research resultKidney-tonifying herbs can promote the osteoblast proliferation and differentiation, improve its morphous structure, inhibit the apotosis of the osteoblast indued by the glucocorticoid, enhance the expression of certain functional protein in the apotosis cells.For these reasons, SANFH can be cured, the method of promoting blood circulation to remove meridian obstruction being used in the process of treating SANFH, at meantime, we should use the method of tonifying the kidney.In this way, superiority can be seen in the evaluation in relieveing pain, improving fuction, adding the joint range of motion.