Effects Of Glucocorticoid On The Function Of Microvascular Endothelial Cells In Human Femoral

BackgroundThe pathogenesis of glucocoticoid-induced osteonecrosis of femoral head remains unclear. Recent researchs suggested that it was closely associated with the injury of bone microvascular endothelial cells. But none of the existing related study use BMECs to do basic research of glucocoticoid-induced osteonecrosis of femoral head. Furthermore, early BMECs were isolated from bone marrow aspirated from iliac crest for study hematological disease, which can’t truly represent endothelial cells lining the interior wall of blood vessels in bone. Therefore, it make more sense to isolate and culture BMECs from femoral head as well as use the cells to study glucocoticoid-induced osteonecrosis of femoral head.ObjectiveThe first aim of this study is to investigate the method for isolation and culture of BMECs of human femoral head, and to establish BMEC lines of human femoral head as well as to provide cell source for later study. The second aim of this study is to investigate the functional changes of BMECs from human femoral head injured by glucocorticoid using microarrays and validated by Quantitative real-time PCR (qRT-PCR).MethodsStudy I.30 femoral heads without pathologic change from patients of 4 males and 26 females were Tesected during hip replacement with the mean age of 71.2 years(range,38-92years). Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion. Cell was isolated by using the methods of enzymic digestion and density gradient centrifugation, purified by differiential attachment, complete endothelial cell culture medium culturing cell, vascular endothelial growth factor (VEGF) and heparin stimulating growth of endothelial cells. Cells were characterized by immunofluorescence analysis for vWF,CD31 and VE-Cadherin as well as VCAM-1.Study II. Based on previous research results in our laboratory, BMECs at passage 3 were cultured in endothelial cell medium containing O.lmg/ml and 0.3mg/ml of hydrocortisone in experimental group to establish glucocorticoid-injured model of BMECs. BMECs in control group were cultured in only endothelial cell medium. Cells were observed at 6,12,18,24 hours using phase contrast microscope after being incubated with hydrocortisone. The differential expression profiles between BMECs injury model and BMECs in control group were tested by mRNA microarrays, the positive genes related to vasoconstriction and vasodilation, coagulation, fibrinolysis and angiogenesis were chosen for qPCR validation thereafter.ResultsStudy I. Observed by phase contrast microscope, the number of cells was positively correlated with patients’ age after 24 hours in primary culture. The older patients were, the less cells numbered.4 to 5 days later, primary cells appeared short spindle, polygon shaped and cobblestone-like morphology. After 7-10 days, primary cells proliferated densely, cell became fusion, arranged in swirl, and contact inhibition appeared significantly. Immunofluorescence staining revealed that the cells were 100% positive for vWF and CD31, and near 100% positive for VE-Cadherin and VCAM-1. it showed that the cells cultured were BMECs.Study Ⅱ. Compared with control group, cells treated with 0.1mg/ml of hydrocortisone shrank and turned small. After 12-24 hours of incubated with 0.1mg/ml of hydrocortisone, the proliferation of cells was inhibited, cell density decreased with poor cell status. However, apoptosis were detected in mostly BMECs treated with 0.3mg/mL hydrocortisone. Cell in control group had a better cellular growth behavior, reaching confluence gradually. So BMECs treated with 0.1mg/ml of hydrocortisone were selected for mRNA microarray detection.519 differential expression genes were found in mRNA microarrays, among which there were 337 genes upregulated,182 genes downregulated. The genes related to vasoconstriction and vasodilation, coagulation, fibrinolysis and angiogenesis such as ET-1 receptor, angiotensin Ⅱ receptor, ICAM-1, PAI-1 were upregulated, and genes of NOS, ET-1, PGI2 synthase, PGI2 receptor, VEGF, PGE synthase, PGE receptor were downregulated. The results of qPCR Validation were consistent with the findings of mRNA microarrays。ConclusionStudy Ⅰ. It was a simple, steady, effective method with good reproducibility, by which highly purified human BMECs can be obtained.Study Ⅱ. Glucocorticoids promote BMECs to express vasoconstrictors, procoagulant factors and related receptor, decreased the expression of the vasodilators and the corresponding receptors.