The Study Of Mechanisms Of Corticosteroid Induced Human Bone Microvascular Endothelial Cell (HBMECs) Injury And Selection For Protecting Drugs

Corticosteroid-induced osteonecrosis of femoral head is one of most causes of nontraumatic osteonecrosis of femoral head in china. Severe acute respiratory syndrome (SARS) patients had been treated with varied dosage of steroids. Due to high dosage of steroids, about one-third SARS patients had occurred osteonecrosis in Beiing. Vascular endothelium damage, stasis of blood stream and blood hypercoagulabale state are the three main causes which lead to osteonecrosis. Therefore, it has important significance to investigate the mechanisms of corticosteroid s induced human bone microvascular endothelial cell (HBMECs) injury and selection for protecting drugs for the intervention therapy of corticosteroid-induced osteonecrosis of femoral head.Objective To isolate isolate and purify human bone microvascular endothelial cells (HBMECs); to establish the model of human bone microvascular endothelial cells (HBMECs) injury induced by glucocorticosteroid; to select effective protecting drugs with the cell model.Methods HBMECs were isolated and purified through immunomagnetic beads with UEA-1, cultured in vitro. The model of HBMECs injury induced by glucocorticosteroid was established. The cytotoxicity of HBMECs was measured by MTT assay, trypan blue staining and TUNEL. The cytotoxicity of HBMECs was measured after incubation with SARS patient'serum. The expression of SARS-CoV receptors ACE2 and CD209L in HBMECs were analysed by the RT-PCR, western-blot and immunocytochemistry respectively. Moreover, the cytotoxicity of HBMECs induced by glucocorticosteroid was analysed in the presence of Prosglandin E1, 7643(an extract from Chinese medicine, Danshen Root), Nadroparin, Enoxaparin, Danshen Root, Ligustrazine, Garlicin respectively.Results HBMECs purified by immunomagnetic beads with UEA-1 have possed the characters of monolayer growth and contact inhibition; Theses cells expressed the molecule marks of VWF, CD31,CD34, and intaked LDL by immune fluorescein stain; After incubation with glucocorticosteroid, the injury of HBMECs was induced with dose dependent manner and time dependent manne, and the results indicated that the injuy is necrosis by MTT assay and trypan blue staining. No obvious cytotoxicity of HBMECs was found after incubation with SARS patient'serum, but SARS patient'serum can increase the sensitiveness of glucocorticosteroid damaging HBMECs. By RT-PCR, Western blot and immunocytochemistry, the both receptors of SARS CoV, ACE2 and CD209L were found to express on HBMECs. With the cell injury model, we found these drugs(PGE1, 764-3, Danshen Root, Ligustrazine) can reduce the cytotoxicity of HBMECs induced by glucocorticosteroid significantly.Conclusion Glucocorticosteroid can damage HBMECs directly in vitro and necrosis is the main mechanism. SARS patient'serum can increase the sensitiveness of glucocorticosteroid damaging HBMECs. PGE1, 764-3, Danshen Root, Ligustrazine, exhibited significant protective effect for glucocorticosteroid induced HBMECs cytotoxicity. DECREASED PROLIFERATION ABILITY OF MESENCHYMAL STEM CELL IN CORTICOSTEROID-INDUCED OSTEONECROSIS OF FEMORAL HEADBackground: The ability of self repair in the patients of corticosteroid-induced osteonecrosis of femoral head is limited, and it has been suggested that the cause is likely relevant to the decreased cell population and the poor proliferation activity of mesenchymal stem cells(MSCs)in the region of femoral head neck or femoral metaphysis of these patients. Therefore, it has important significance to detect the proliferation and differentiation ability of MSCs isolated from the bone marrow in patients with steroid-induced osteonecrosis of femoral head. It can provide rational evidences for treatment of autologous bone marrow stem cell transplant for osteonecrosis of femoral headMethods: The bone marrow (3ml～5ml) was collected from the proximal femur of having surgical therapy for either steroid-induced osteonecrosis of the femoral head(Severe Acute Respiratory Syndrome-post-osteonecrosis, case group) or new femoral neck(control group). Case group: surgical therapy including compacting bone graft through femoral head and neck windowing, male 7 cases, female 11 cases; Control group: male 3 cases, female 8 cases who had not developed osteonecrosis of femoral head nor received corticoid treatment. They were diagnosed as new fracture of femoral neck and intended to undergo hip replacement served as controls. Their age ranged from 55 to 85 years with a mean of 60 years. The mesenchymal stem cells were isolated by density gradient centrifugation, and then selected by the adhesive method. The method of MTT was employed to evaluate the level of proliferation. The parameters regarding the variation of the cell cycle were detected with the flow cytometry(FCM)Results: The results of MTT assay indicated that the cells obtained from the patients with steroid-induced osteonecrosis of femoral head showed reduced proliferation ability compared with the cells obtained from the patients with femoral neck fracture. The percentage of cells at the(S＋G2/M) phase was decreased significantly (P＜0.05) in Case Group.Conclusion: The decreased proliferation ability of mesenchymal stem cell may play a role in the low repair capacity of steroid-induced osteonecrosis of femoral head. The altered function of MSCs can be responsible for the pathogenesis of osteonecrosis.