Study or the effects of estrogen on voltage-dependent ionic channels in osteoclast cells

基础实验：取新生Wistar大鼠(1~2日龄)腿部长骨获取破骨细胞。取下胫骨和股骨，放入培养液(最低必需培养液；Gibco BRL)中，切碎。用大口径吸管轻轻搅拌悬浮液，破骨细胞从骨表面分离。这个悬浮物被种在玻璃封盖上。使用一个含有三个或更多细胞核的细胞进行全细胞记录。这些细胞被确定为破骨细胞，因为当细胞在牙本质切片上培养24小时时，这些细胞抗酒石酸酸性磷酸酶(TRAP)染色阳性，并形成吸收坑。在以改良泰氏液为细胞内液，高钾溶液为移液的条件下，全细胞电压钳可记录到破骨细胞内向整流型钾电流。L7β-雌二醇在1μM以上剂量依赖性地抑制这种内向整流型钾电流。这一结果表明，雌二醇通过抑制破骨细胞内向复位型钾电流而抑制破骨细胞对骨的吸收。临床研究：在卵巢切除妇女中，使用结合马雌激素(CEE；0.625 mg/d)进行激素替代治疗，并用双能X射线骨密度仪计算这些妇女的骨密度。我们发现，在卵巢切除后的最初两年内，仅服用0.625毫克/天的CEE并不能预防急性骨丢失，这表明可能需要额外的治疗来预防骨质疏松。此外，我们还发现，卵巢切除后早期联合应用依普列芬和CEE可抑制骨丢失，并被认为能有效地维持卵巢切除后的骨量。

Basic experiments : Osteoclasts were obtained from the leg long bones of neonatal Wistar rats (1-2 day old). The tibiae and femora were removed and then placed in culture medium (minimum essential medium alpha medium ; Gibco BRL), and finely chopped. The suspension was gently agitated with a wide bore pipette and osteoclasts were dissociated from the bone surface. This suspension was seeded on glass coverslips. A cell with three or more nuclei was used for the whole-cell recordings. These cells were determined to be osteoclasts because such cells stained positive for tartrate-resistant acid phosphatase (TRAP) and formed resorption pits when the cells were cultured on dentine slices for 24 hr. In the condition using modified Tyrode solution as an intracellular solution, and high-potassium solution as a pipette solution, inwardly rectifer type potassium current could be recorded in osteoclasts by whole-cell voltage clamp. l7 β-Estradiol at the concentration above 1μM inhibited this inwardly rectifer type potassium current in dose-dependent manner. This result suggests that estradiol inhibits bone absorption by osteoclasts by the inhibition of inwardly reclifer type potassium current in osteoclast cells.Clinical research : In oophorectomized women, hormone replacement therapy using conjugated equine estrogen (CEE ; 0.625mg/day) was done, and the bone mineral density was calculated by means of dual energy X-ray absorptiometry in these women. We found that in the initial 2-year period after oophorectomy, 0.625 mg/day of CEE alone could not prevent acute bone loss suggesting that additional therapy for the prevention of osteoporosis may be needed. Furthermore we found that concomitant use of ipriflavone with CEE from an early stage after oophorectomy inhibited bone loss and was considered to be effective in maintaining bone mass after oophorectomy.