NMDA接受器表現降低參與卵巢切除所引發的骨質稀少

Suppression of NMDA receptor participates in the mechanism of ovariectomy induced osteopenia in rats

鄭詠如(Yung-Ru Jeng)

指導教授 ： 許勤

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

長期雌性素減少所引起的骨質流失已被廣泛研究，而主要增加蝕骨細胞生成。先前研究指出，卵巢切除後大腦海馬迴區之麩胺酸鹽主要接受器N-methyl-D-aspartate receptor(NMDAR)表現減少。最近研究確認骨細胞上含有麩胺酸鹽轉運因子及NMDA接受器；而本實驗室先前的研究也指出廢用導致骨質流失的動物模式中NMDAR表現降低，推測麩胺酸鹽可能在骨骼扮演重要角色。然而，是否NMDA接受器參與在卵巢切除所引起的骨質減少是尚未了解的。因此本研究主要探討NMDA接受器在卵巢切除導致骨質減少所扮演的角色。使用卵巢切除引起骨質減少之動物模式，雙側卵巢切除後3個月以DEXA（Dual energy X-ray absorptiometry）測量骨質密度、量化骨小樑之面積來確認骨質稀少之動物模式的建立。偵測骨形成標誌(core binding factor ??-1 (cbfa1/Runx2), osterix, type I collagen, alkaline phosphatase (ALP), osteocalcin)，骨溶蝕標誌(tartrate-Resistant Acid Phosphatase (TRAP))及蝕骨細胞分化之調節因子(osteoprotegerin (OPG)、 nuclear factor-?羠 ligand (RANKL))以RT-PCR量化RNA層面的變化。利用RT-PCR、西方點墨分析法和免疫組織染色，確認卵巢切除導至骨質稀少之老鼠NMDA接受器次單位NR1及NR2D基因及蛋白質表現與對照組之差異，了解NMDA接受器是否參與骨質稀少之機制探討。活體外實驗使用NMDA接受器非競爭性拮抗劑，MK-801，作用在骨髓間質細胞和成骨細胞，來探討NMDA接受器對於成骨細胞生成，蝕骨細胞分化因子及蝕骨細胞新生之影響。實驗結果顯示：1）卵巢切除組在股骨組織萃取中TRAP mRNA的表現顯著上升；cbfa1/Runx2、osterix mRNA的表現顯著降低，而osteocalcin是顯著上升的;OPG顯著下降，RANKL顯著上升。2）卵巢切除後，在股骨組織萃取中卵巢切除組NR1之mRNA及蛋白質的表現顯著低於對照組；此外免疫組織染色的結果顯示，卵巢切除組之NR1染色在骨髓細胞、成骨細胞和蝕骨細胞顯著下降。3)活體外實驗顯示，MK-801，在骨髓間質細胞抑制cbfa1/Runx2、osterix及促進RANKL基因表現；在成骨細胞抑制OPG基因表現；其上清液培養促進蝕股細胞新生。而由活體內及活體外結果推測在卵巢切除的情況下，可能透過降低NMDA接受器的表現，抑制成骨細胞之新生和促進蝕骨細胞新生，因而導致骨質稀少。

關鍵字

蝕骨細胞 ； 成骨細胞 ； 卵巢切除 ； 骨髓間質細胞

並列摘要

Bone loss caused by the increase of osteoclastogenesis after long-term estrogen deficiency has been extensively studied. Previous report indicated that N-methyl-D-aspartate (NMDA) receptor, the major subtype of glutamate receptors, was decreased in hippocampus after ovariectomy (Ovx). The recent identification of the NMDA receptor in bone cells and previous results showed that suppression of NMDA receptor in disuse osteopenia, and therefore suggested a role for glutamate in the skeleton. However, whether NMDA receptor is involved in bone loss caused by Ovx remains unknown. Therefore, the present study was designed to investigate the role of NMDA receptor in Ovx-induced osteopenia. The rat osteopenia model was established by bilateral ovariectomy. Three months after Ovx, osteopenia was confirmed by bone mineral density and trabecular bone volume. The femur mRNA expressions of bone formation markers, core binding factor ??-1 (cbfa1/Runx2), osterix, type I collagen, alkaline phosphatase (ALP), osteocalcin；bone resorption marker, tartrate-resistant acid phosphatase (TRAP), and osteclast differentiation factor, osteoprotegerin (OPG) and receptor activator of NF?羠 ligand (RANKL) were measured by RT-PCR. The femur mRNA, protein content and cellular distribution of the subunit proteins of NMDA receptor, NR1 and NR2D, were evaluated by RT-PCR, western blot analysis and immunohistochemistry, respectively. The mediation of NMDA receptor in osteoblastogenesis or osteoclast differentiation was evaluated by blocking NMDA receptor with MK-801, a non-competitive NMDA receptor antagonist. The results showed that: (1) The mRNA expressions of cbfa1/Runx2, osterix and ratio OPG and RANKL mRNA were significantly decreased, while the mRNA expressions osteocalcin and TRAP were increased after Ovx. (2) The mRNA expressions and protein content of NR1 was significantly lowered in the femur bone tissue of Ovx group than in control group. In addition, the decrease of NR1 immuno-reactivity was prominent in bone marrow cells, osteoblasts and osteoclasts. (3) In-vitro showed that the mRNA expressions of cbfa1/Runx2 and osterix in bone marrow stroma cells(BMSC) and mRNA expression of OPG in osteoblasts were inhibited, while the mRNA expression of RANKL in bone marrow stroma cells was enhanced after MK-801 treatment；culture supernatant derived from BMSC and osteoblasts treated with MK-801 enhancing osteoclastogenesis. These results suggested that of NMDA receptor may participate in the Ovx-induced osteopenia via inhibiting osteoblastogenesis and enhancing osteoclastogenesis.

並列關鍵字

NMDAR(N-methyl-D-aspartate receptor) ； MK-801 ； ovariectomy ； osteoblast ； bone marrow stroma cell

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