- 學位論文
脈衝式電磁場刺激對蝕骨細胞之生物效應: 其在 骨質疏鬆防治上之臨床應用
Biological Effects of Pulsed Electromagnetic Fields Stimulation on Osteoclasts: Its Clinical Relevance to Osteoporosis Management
摘要
生物電磁學(Bioelectromagnetics)是一門研究電磁場與生物體間交互作用的新興科學。在所有的生物體器官或組織內，例如骨頭、神經、軟骨、肌肉….等都存在有電性現象。因此，這些組織亦都會受到外加磁場或電磁場之影響。事實上，有愈來愈多的證據證實了環境當中所存在頻率低於300赫茲之電場或磁場可以影響生物功能；但是其作用機轉到目前為止尚未明瞭。即使電磁場在生物體的影響機轉尚處於混沌且爭論的階段，並且因此阻礙了生物電磁學在醫學領域的應用；一些具有特殊參數之時變電磁場，在過去三十年當中已經成功應用在相當多具有挑戰性之醫學問題上，尤其是在小孩、大人身上所發生之骨折不癒合。另外，在慢性皮膚傷口癒合上電磁場亦扮演了促進之角色。最近由於已開發國家一般人口之年齡大幅提昇，另外一種骨頭疾病-骨質疏鬆症(Osteoporosis)的高發生率亦開始引起生物電磁學家之注意。在相關動物及臨床試驗中，電磁場刺激-尤其是脈衝式電磁場刺激，又再一次證實了其在骨質疏鬆症上之防治效力。 為了試圖要澄清及證實脈衝式電磁場在骨質疏鬆上防治效力，本論文即設計並執行了一系列體外細胞培養實驗。這篇論文的主要研究目的就是想要從細胞層次來研究脈衝式電磁場是如何對骨組織疾病；特別是骨質疏鬆症，產生防治效果。並且希望能研究並找岀可以合理解釋脈衝式電磁場作用效力之潛在可能機轉。在接下的段落中，我們將以個別章節之模式，來逐一簡介這篇論文當中一系列不同但卻又息息相關之實驗。在第二章中我們首先發展出一套脈衝式電磁場刺激器，並且採用赫模霍茲線圈(Helmholtz coils)作為刺激已接受雙側卵巢切除術成熟母鼠之刺激線圈。全程刺激時間為一小時。組織型態學分析以及放射性免疫分析法，分別用來評估脈衝式電磁場刺激對卵巢切除所誘發之骨質流失的影響；以及對其血清中前列腺素-E2 (Prostaglandin-E2; PGE2)濃度變化之影響。在本篇實驗中，我們得知每天8小時之脈衝式電磁場刺激可以有效防止由雌激素缺乏所導致之骨質疏鬆；而前列腺素-E2可能是其中之媒介物。 極低頻脈衝式電磁場刺激對經由大鼠骨髓細胞體外培養模式所形成蝕骨細胞之影響將在第四章中探討。在本實驗當中，成熟大鼠骨髓之細胞在取出後即接受不同感應電場強度(4.8 μv/cm, 8.7 μv/cm, and 12.2 μv/cm; Chamber slide; 峰值)以及不同刺激時間之(0.5 h/day, 2 h/day, and 8 h/day)脈衝式電磁場刺激；連續刺激9天。在實驗當中，分別於第5、7、9天收取上清液並利用酵素免疫分析法分析腫瘤壞死因子-α (Tumor necrosis factor-α; TNF-α)、介白素-1β (Interleukin-1β; IL-1β)、以及前列腺素-E2。由結果得知脈衝式電磁場可以影響蝕骨細胞形成，且腫瘤壞死因子-α以及介白素-1β可能是其作用媒介物。 而在第五章的實驗當中，最主要是想利用從卵巢切除不同天數後之大鼠骨髓細胞來建立體外蝕骨細胞培養模式；之所以這樣做是想要更進一步模擬第二章所做過之體內動物研究模式。在這一部份之實驗我們採取由第四章所找出之最佳刺激參數(4.8 μv/cm, 2 h/day)來進行為期9天之刺激。結果發現接受此參數刺激後之蝕骨細胞形成個數明顯受到抑制：分別為降低55% (雙側卵巢切除術後4天)以及降低43% (雙側卵巢切除術後7天)。另外，細胞激素之分析結果亦顯示接受脈衝式電磁場刺激後之腫瘤壞死因子-α、介白素-1β、以及介白素-6有明顯合成受到抑制之現象發生。這部份實驗驗證了即使所取得的大鼠骨髓細胞是由卵巢切除後之動物而來，脈衝式電磁場刺激仍然有影響其分化為蝕骨細胞之效力。而腫瘤壞死因子-α、介白素-1β、以及介白素-6在脈衝式電磁場刺激過程中扮演了相當重要之角色。 為了能夠更加澄清脈衝式電磁場刺激對骨質疏鬆生物效應之潛在機轉，我們在第六章當中從另外一個角度-細胞凋亡(Apoptosis)切入；並設計一個實驗來探討脈衝式電磁場刺激是否對蝕骨細胞具有凋亡促進之效力。在此研究中由於所採用之培養皿為圓形結構，因此理論計算出來之感應電場強度約為3.0 μv/cm。當體外造骨細胞與大鼠骨髓細胞，所共同培養得到之成熟蝕骨細胞接受酵素分離後，即接受此一強度之脈衝式電磁場刺激。刺激時間分別為1、8、16小時，而分別於刺激完後之48小時後觀察蝕骨細胞凋亡比率。研究結果發現連續接受脈衝式電磁場刺激8 和16小時後之蝕骨細胞凋亡比率(105%, 30%)均有顯著上之提昇；意即有促進蝕骨細胞凋亡之現象發生。此結果因此可作為脈衝式電磁場刺激在體內動物實驗所觀察到骨質流失防治之可能原因之一。 第七章所設計的實驗與第四章有相似之處，然而我們主要是想藉由分析與蝕骨細胞分化過程當中，具有相當密切關係之細胞激素如osteoprotegerin (OPG)、receptor activator of NFκB-ligand (RANKL)、巨噬細胞-群落刺激因子(Macrophage colony-stimulating factor; M-CSF)等之合成是否會受到外加脈衝式電磁場刺激之調控。結果發現到，脈衝式電磁場刺激影響蝕骨細胞分化之過程，與這些因子均有相當密切之關係。此外，由於脈衝式電磁場之生物實驗在體內動物及體外細胞培養實驗上，所誘發出來之感應電場強度不一樣；因此在第三章當中回顧了相關電磁場理論基礎之文獻以及書籍。在本章當中，體外細胞培養實驗之刺激線圈系統、磁場與電場之量測與理論計算、實驗培養皿之幾何結構對感應電場強度之影響、以及一些在本研究當中實際感應電場強度之計算例子；均會在此章節有一詳盡之介紹與討論。 由本論文所介紹之一系列實驗結果當中，可以確定的一件事實就是外加電磁場能量確實可以對蝕骨細胞之分化以及凋亡調控產生不一樣且重要之生物效應。此外，這些生物效應並不是經由其如此低強度所產生之熱效應所達成。根據本論文所產生出之一系列研究成果，我們可以推論脈衝式電磁場刺激之所以能夠防治骨質疏鬆症；可能一部份是經由調控骨組織中之蝕骨細胞而達成。這項推論在未來有可能成為脈衝式電磁場刺激，在臨床骨科醫師防治骨質疏鬆症以及其他醫學科學領域應用上，被採用作為新輔助療法模式之基石。
並列摘要
Bioelectromagnetics (BEM) is the emerging science that studies how living organisms interact with electromagnetic (EM) fields. Electrical phenomena are found in all living organisms such as bone, nerve, cartilage, muscle…etc. Consequently, they can be influenced by external magnetic and EM fields as well. In fact, there is growing evidence that environmental electric and magnetic fields in the extremely low frequency (ELF) band below 300 Hz can influence biological functions by mechanisms that are only poorly understood at the present time. In spite of the ambiguous and controversial mechanistic issues, which have been confused and hamper the advance and application of BEM in medical community, selected, time-varying electric and magnetic fields have played an increasingly successful role in the care of several challenging medical problems, mainly fractures that have failed to heal in children and adults and chronic skin wounds during the past three decades. Recently, due to the prolonged lifespan of average population, the prevalence of another kind of bone disease-osteoporosis has caught the attention of BEM scientists. Again, EMF, especially pulsed electromagnetic fields (PEMF) was proved to be effective on the management of osteoporosis. With an attempt to clarify and substantiate the efficacy of PEMF on osteoporosis management, a series of in vitro experiments were designed and performed this dissertation. The primary objectives of this dissertation are to investigate the interactions between PEMF and bone tissues at the cellular level, and the underlying mechanisms that lead to the explanation for the PEMF efficacy. In succeeding paragraphs we present the synopsis of different but connected experiments carried out in this thesis in terms of chapters. In Chapter 2 we developed the PEMF stimulators and employed Helmholtz coils to stimulate female rats that have been ovariectomized bilaterally. The total stimulation time was one month. Histomorphometric analyses and radioimmunoassay (RIA) were used to evaluate the effects of PEMF on ovariectomy-induced trabecular bone loss and serum PGE2 concentration. These experiments demonstrated that PEMF may be useful in the prevention of osteoporosis resulted from estrogen deficiency, and PGE2 might relate to this preventive effects. The effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)2D3, were examined in Chapter 4. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8 μv/cm, 8.7 μv/cm, and 12.2 μv/cm; peak value) and stimulation times (0.5 h/day, 2 h/day, and 8 h/day). During the experiments, cytokines such as tumor necrosis factor-α (TNF-α), interleukin -1β (IL-1β), and prostaglandin-E2 (PGE2) were assayed by using enzyme-linked immunosorbent assay (ELISA). Results showed that PEMF can affect osteoclastogenesis and TNF-α and IL-1β might involve in this process. The purpose of Chapter 5 was to examine the effects of a specific PEMF stimulation on osteoclast formation in bone marrow cells from ovariectomized rats and to determine if the signal modulates the production of cytokines associated with osteoclast formation. Under the stimulation of 4.8 μv/cm electric field intensity, the PEMF signal caused significant reductions in osteoclast formation in both Subgroups I (4 days after surgery) (-55%) and II (7 days after surgery) (-43%). Tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in PEMF group of Subgroup I were significantly reduced at 5, 7, and 9 days as compared to OVX group. The results found in this study suggest that osteoclastogenesis can be inhibited by PEMF stimulation, putatively due to a concomitant decrease in local factor production. In order to further clarify the underlying mechanism of PEMF bioeffects on osteoporosis, the effect of pulsed electromagnetic fields (PEMF) on induction of osteoclasts apoptosis was investigate in Chapter 6. A statistically significant increase of apoptotic rate in osteoclasts (48 hours after isolation) was found when exposed to 7.5 Hz PEMF with induced electric fields intensity of 3.0 μv/cm for 8 (105%, p<0.001) (PEMF-8) and 16 (30%, p<0.05) (PEMF-16) hours. However, exposure of osteoclasts to PEMF for only 1 hour (PEMF-1) showed no statistically significant differences when compared with controls. These findings suggest that PEMF have the ability to induce apoptosis of osteoclasts derived from primary osteoblasts and bone marrow cells cocultures. This in vitro study, therefore, could be considered as groundwork for in vivo PEMF applications on some osteoclasts-associated bone diseases. Chapter 7 described an experiment that is similar to Chapter 4. However, cytokines closely related to osteoclastogenesis such as osteoprotegerin (OPG), receptor activator of NFκB-ligand (RANKL), macrophage colony-stimulating factor (M-CSF) were determined in order to further clarify the underlying mechanism. The results found in this study suggest that osteoclastogenesis can be modulated by PEMF stimulation, putatively due to concomitant variations in local factors production. Moreover, due to the induced electric field intensity in in vivo and in vitro situations was different, some related literatures were reviewed and presented in Chapter 3. In this chapter, exposure systems for in vitro studies, measurement of magnetic fields, measurement of electric fields, effects of experimental geometry of culture dishes, and some practically calculated examples of induced electric field intensity in our experiment were discussed. The conclusion that electromagnetic energy can produce varied and nontrivial biological effects on osteoclasts differentiation and apoptosis modulation is inescapable based on our findings. Furthermore, the evidence that such interactions can occur well below the thermal level is similarly inescapable. With these findings of experiments, we can infer that the effects of PEMF stimulation on osteoporosis management might partially due to its modulating ability on osteoclasts. This inference might in the future become the cornerstone for PEMF stimulation to be adopted as an adjunct alternative not only for osteoporosis management in clinical orthopedics but also in other medical frontiers.