利用骨髓細胞培養模式探討單脈衝電磁場刺激對去卵巢後大白鼠蝕骨細胞形成的影響

An in Vitro Study of Effects of the Single Pulsed Electromagnetic Fields Stimulation on Osteoclast Formation from Bone Marrow Cells of Ovariectomized Rats

游晏欣(Yen-Shin Yu)

指導教授 ： 張恆雄

中原大學/工學院/醫學工程研究所/碩士(2001年)

https://doi.org/10.6840/cycu200100410

摘要

摘要

本研究旨在探討雙側卵巢切除後大白鼠之蝕骨細胞變化情形及此蝕骨細胞接受單脈衝電磁場刺激後之反應。在動物實驗方面已經證實：單脈衝式電磁場能有效地控制卵巢切除後大白鼠之骨質流失情形。而體外細胞培養方面也已找到特定強度單脈衝電磁場刺激骨髓細胞，能抑制蝕骨細胞合成。為了要更進一步探討電磁場刺激抑制骨質流失的內在機轉，因此本研究將以大白鼠之雙側卵巢切除為模式，取出長骨骨髓細胞做體外培養，藉此探討特定參數單脈衝電磁場刺激是否經由對骨髓中的蝕骨細胞形成，產生抑制作用，而達到控制骨質流失的目的。
本研究採用6~11個月大，成熟雌性Wistar大白鼠為實驗對象。分成完整組（Int.）、假性卵巢切除組（Sham）、雙側卵巢切除組（Ovx.）及雙側卵巢切除加單脈衝電磁場刺激組（Ovx.+Sti.）。在卵巢切除後第4、7、及14天犧牲，分別取股骨與脛骨做為骨髓細胞之來源，比較其差異。結果卵巢切除後14天模式下，雙側卵巢切除組蝕骨細胞數目明顯較完整組少且有統計上的差異。而卵巢切除後7天模式下所得到的蝕骨細胞個數及平均核數在卵巢切除組與完整組間沒有差異。卵巢切除後4天模式下，卵巢切除組之平均核數較完整組多且有統計上的差異。
用來刺激的電磁場參數為：單脈衝波形、頻率7.5Hz、誘發之電場強度為2mv/cm。細胞分盤後放入培養箱培養一天，再開始刺激。刺激時間為1小時/天，共刺激9天。刺激結束後，進行評估。評估的項目包括：1.抗酒石酸鹽酸性磷酸@染色（TRAP Stain），以確認蝕骨細胞，並計數細胞的數目。2.置入骨薄片，以觀察骨吸收的作用。3.介白質-6（IL-6）、介白質-1β（IL-1β）與腫瘤壞死因子-α（TNF-α）的濃度分析，以探討這些細胞激素與蝕骨細胞生成及活性間的關係。
結果證明卵巢切除14、7及4天後之犧牲模式，取得母鼠之骨髓細胞做體外培養並接受特定強度電磁場刺激，證明電磁場刺激均可有效抑制蝕骨細胞的形成，且可有效抑制IL-1β、IL-6及TNF-α等細胞激素的分泌。

關鍵字

骨髓細胞 ； 卵巢切除術 ； 蝕骨細胞 ； 單脈衝電磁場

並列摘要

Abstract
The purposes of this study are intended to investigate the changes of osteoclast formation from in vitro bone marrow culture cells of ovariectomized rats and the effects of single pulsed electromagnetic fields (PEMF) stimulation on osteoclast formation and activity. It has been confirmed not only on animal model that PEMF stimulation can decrease the ovariectomized rats bone loss effectively but also on in vitro cell culture model that PEMF stimulation can inhibit osteoclast proliferation. In order to clarify the mechanisms of PEMF on osteoporosis, bone marrow cells derived from both femora and tibiae of ovariectomied rats are cultured in vitro to investigate whether osteoclast proliferation is inhibited by PEMF or not.
This study used 6~11-month mature female Wistar rats as experimental subjects. The rats were randomly divided into four groups: 1) Intact group (Int.); 2) Sham ovariectomy group (Sham); 3) Bilateral ovariectomy group (Ovx.); 4) Bilateral ovariectomy + PEMF (Ovx. + Sti.). Int., Sham, Ovx., and Ovx.+Sti. were sacrificed at 14, 7, and 4 days after ovariectomy. Bone marrow cells were acquired from both femora and tibiae of each group. After 24 hours culture in incubator with 37oC and 5% CO2 surroundings, the cells were stimulated by PEMF with induced electric fields of 2mv/cm and the stimulation protocol was 1 hr/day for 9 days. To investigate whether the same PEMF parameters have effects on bone marrow cells culture of ovariectomized rats.
At the end of stimulation, the tartrate-resistant acid phosphonatase (TRAP) staining method was applied to validate and enumerate osteoclast cell and porcine slice were used to reconfirm the authentication of osteoclast as well as the observation of bone resorption activity. Enzyme-link immunosorbent assay (ELISA) was employed to measure the concentrations of TNF-α, IL-1β, and IL-6 to research the relationship between these cytokines and osteoclasts formation.
The results showed that：(1) The osteoclastogenic potential from bone marrow cells was different among 14, 7, and 4 days after ovariectomy; (2) PEMF stimulation could inhibit the osteoclast proliferation from the in vitro bone marrow cell culture model of ovariectomized rats; (3) PEMF stimulation could decrease the concentrations of IL-6, TNF-α, and IL-1β from the cell culture supernatant.

並列關鍵字

bone ； ovariectomy ； Single pulsed electromagnetic fields

參考文獻

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