血小板和内皮细胞源性微粒与兔激素性股骨头缺血坏死发生机制的实验研究

Platelet and endothelial cell-derived microparticles in steroid-induced osteonecrosis of the femoral head of rabbit model

目的探讨激素诱发血液高凝易栓导致骨缺血坏死发病过程中血小板和内皮细胞源性膜微粒数量变化。方法选取健康成年日本大白兔44只,随机分为两组,实验组28只,按20 mg/kg臀肌注射甲泼尼龙1次;对照组16只按相同剂量臀肌注射生理盐水1次。注射前、注射后1、2、4、8周各抽静脉全血,测定血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTF)、抗凝血酶-Ⅲ(AT-Ⅲ)。流式细胞仪对 CD31、CD42a、CD54、CD62E 进行定量、定性测定。试验组、对照组分别于1、2、4、8周各处死6、4只取双侧股骨头组织学检查,HE 和微血栓染色。结果 1周时试验组凝血功能增强,CD31、CD42a、CD54、CD62E微粒阳性表达并数量较对照组显著增高(t=0.008,P<0.05),并持续至8周;组织学1周时,骨髓水肿、细胞坏死碎片、出血,出现空骨陷窝,微血栓染色阳性;2～8周时微血栓数量增加,骨坏死明显,骨髓细胞脂肪变性、脂肪细胞直径增大、脂肪组织面积比增加。结论应用大剂量糖皮质激素后兔体内血小板和内皮细胞源性膜微粒数量增加,具前凝血潜能的微粒数量的增加可能是大剂量糖皮质激素诱发血液高凝易栓、导致股骨头缺血坏死发生的原因之一。抑制微粒的产生或许有助于预防或避免激素诱发股骨头缺血坏死的发生。

Objective To explore the relationship between the alterations of platelet-derived microparticles （ PMPs ） and endothelial cell-derived microparticles （ EMPs ） and steroid-induced osteonecrosis of the femoral head. Methods Forty-four adult Japanese white rabbits were randomly divided into 2 groups： experimental group （n = 44 ）, injected intramuscularly once with 20 mg/kg of methylprednisolone acetate, and control group （ n = 28） injected with normal saline of the same dose. Blood samples were collected from the auricular vein while the animals were in a fasting state in early morning prior to experiment （week 0）, and 2, 4, and 8 weeks after the injection. The levels of prothrombin time （PT）, activated partial prothrombin time （APTT）, and anti-thrombinase-Ⅲ （AT-Ⅲ） were examined. Plasma was ultra-centrifuged and the circulating platelet and EMPs were isolated to undergo flow cytometry to detect the amounts of CD31, CD42a, CD54, and CD62E. 1, 2, 4, and 8 week after the injection 6 rabbits from the experimental group and 4 rabbits from the control group were sacrificed respectively and their femoral heads were taken out to undergo histopathologic examination with HE staining and microthrombus and elastic fiber staining. Results The PT, APTT, and AT-Ⅲ levels increased significantly 1 to 8 weeks after the injection. The numbers of EMPs and PMPs were also increased markedly after steroid administration. Histopathologic examination demonstrated an accumulation of bone marrow cell debris, presence bone empty lacunae in the trabeculae 1 week after the steroid injection. 2, 4, and 8 weeks after the steroid injection the bone necrosis was remarkable, fatty degeneration of bone marrow cells occurred, the diameter of fat cells increased, and the area ratio of fatty cells became higher. MSB staining was positive. Conclusion High dose glucocorticosteroid increases the levels of PMPs and EMPs that may be related to hypercoagulability and thrombosis in microcirculation and play an important role in steroid-induced osteonecrosis. MP can be considered a true target in the pharmacological control of steroid-induced osteonecrosis.