不同刺激時間單脈衝電磁場對造骨細胞與骨髓細胞共同培養形成之類蝕骨細胞凋亡的影響

Effects of Different Stimulation Time of sPEMF on Apoptosis of Osteoclast-Like Cells Developed from Coculture of Osteoblast Cells and Bone Marrow Cells

蔡明慈(Ming-Tzu Tsai)

指導教授 ： 張恆雄 ； 史中

中原大學/工學院/醫學工程研究所/碩士(2002年)

https://doi.org/10.6840/cycu200200483

摘要

骨質疏鬆症主要是由於體內的骨吸收作用大於骨形成作用而造成。影響骨吸收的因子有許多，其中蝕骨細胞是最直接且強而有力的影響因子。本實驗室已找到特定參數之單脈衝電磁場能有效抑制類蝕骨細胞之生成，因此本研究之目的欲觀察此單脈衝電磁場對類蝕骨細胞凋亡的效應。首先需建立一造骨細胞與骨髓細胞之共同培養系統以形成類蝕骨細胞，並在不同時段分析類蝕骨細胞在接受單脈衝電磁場刺激後凋亡的比例，期許電磁場能達到促進蝕骨細胞凋亡的作用，進而能治療或預防骨質疏鬆症的產生。
本研究採用10～12個月大之成熟雌性Wistar大鼠的骨髓細胞作為此共同培養系統的主要來源，並分別與新生ICR小鼠與新生Wistar大鼠之造骨細胞做共同培養。造骨細胞密度為1x106 cells/6-cm dish，骨髓細胞培養密度分別為2x107、4x107、8x107 cells/6-cm dish。不同鼠種與不同細胞密度配對下共有6種共同培養模式。在為期8天的培養中，每次更換培養液時皆加入10 nM 1α,25(OH)2D3以促進類蝕骨細胞分化、生成。培養第8天時，以0.15% 膠原蛋白酵素進行細胞分離步驟，以得到純化的類蝕骨細胞。此時純化過之類蝕骨細胞即接受不同時間之單脈衝電磁場刺激。細胞被隨機地分為4組：Control（控制組）、PEMF-1（刺激1小時組）、PEMF-8（刺激8小時組）以及PEMF-16（刺激16小時組）。電磁場之參數為：單脈衝波形、頻率7.5 Hz、誘發電場強度2 mV/cm。
待電磁場刺激結束後，再分別於0、24、48、72小時（細胞分離結束時為0小時）以PI染色法進行細胞凋亡之染色分析，觀察類蝕骨細胞的凋亡比例。
在類蝕骨細胞之定性方面：（1）以tartrate-resistant acid phosphatase（TRAP）來定性類蝕骨細胞之酸性磷酸酵素。具TRAP活性且具有3顆核以上的多核細胞才定義為類蝕骨細胞；（2）以掃瞄式電子顯微鏡觀察類蝕骨細胞在豬皮質骨片上產生骨吸收凹陷情形。
實驗結果顯示：（1）新生ICR小鼠的造骨細胞（1x106 cells/6-cm dish）與成熟雌性Wistar大母的骨髓細胞（4x107 cells/6-cm dish）可建立最佳共同培養系統，且具TRAP活性之多核細胞，另外5種配對狀態之細胞則皆無TRAP活性。而在電磁場均勻度的考量下，將直徑6公分培養皿更改為直徑3.5公分培養皿，因此最佳共同培養系統之造骨細胞與骨髓細胞為4x105 cells/3.5-cm dish與2x107 cells/3.5-cm dish。而在此培養模式下仍能形成大量多核且具TRAP活性之類蝕骨細胞，且具骨吸收活性；（2）在細胞凋亡分析上，不論有無接受電磁場刺激，類蝕骨細胞凋亡的比例皆隨著時間的增加而遞增，且在72小時之凋亡比例達到最大（98％）；經由PI染色可觀察類蝕骨細胞之凋亡現象會隨著時間增加而有愈趨明顯之核片段產生；PEMF-1與Control在0～72小時之類蝕骨細胞凋亡比例皆趨近於相同；PEMF-8與PEMF-16在48小時的類蝕骨細胞凋亡比例皆高於Control，而0、24、72小時則與Control趨近於相同。
結果顯示特定參數下之單脈衝電磁場對於誘使類蝕骨細胞凋亡，會因電磁場刺激時間與凋亡分析時間而有所不同。而8小時與16小時之電磁場刺激則可促進類蝕骨細胞在48小時後的凋亡比例增加。

關鍵字

骨髓細胞 ； 單脈衝電磁場 ； 造骨細胞 ； 類蝕骨細胞

並列摘要

Osteoporosis occurs when the activity of bone resorption is larger than the activity of bone formation. The osteoclast is one of the most direct and powerful factors, which is involved in bone resorption. Our lab have been proved that pulsed electromagnetic fields (PEMFs) with specific parameters influence the inhibition of osteoclast differentiation and formation, and this study investigates the effect of PEMF on osteoclast apoptosis. First, a novel coculture system is established to develop osteoclast-like cells (OCLs), which are exposed to PEMFs for different stimulation times, and OCL apoptosis rates are assayed at different times after PEMF exposure. We expect that PEMFs have effects on treatment or prevention of osteoporosis by inducing osteoclast apoptosis.
In this study, bone marrow cells (BMCs) obtained from 10~12 -month-old adult female Wistar rats were the major cells in this coculture system. Osteoblasts (OB) were obtained from new-born ICR mice and new-born Wistar rats, and were individually cocultured with BMCs. OB density was 1x106 cells/6-cm dish, and BMC densities were 2x107, 4x107, and 8x107 cells/6-cm dish, giving a total 6 of types of coculture models. 10 nM 1α, 25(OH)2D3 was added in culture medium in coculture periods to enhance OCL differentiation. On the eighth day, purified OCLs were acquired after isolation by 0.15% collagenase, and isolated OCLs were exposed to PEMFs with different stimulation times. There were four groups: Control, PEMF-1 (exposure for 1 hr ), PEMF-8 (exposure for 8 hr), and PEMF-16 (exposure for 16 hr). The PEMF parameters were: single pulse, 7.5 Hz of frequency, and 2 mV/cm of induced electric field intensity.
Apoptosis rates of OCLs were assayed by PI-stain method individually at 0, 24, 48, and 72 hr after PEMF exposure, where the time point after isolation was defined as 0 hr.
For qualitative analysis of OCLs there were two processes. In one, Tartrate-resistant acid phosphatase (TRAP) was used as a marker of acid phosphatase of OCLs, and TRAP-positive multinucleated (more than 3 nuclei) cells (TRAP-PMCs) were identified as OCLs. In the other, bone resorption pits on the surface of porcine cortical bone slices formed by OCLs were examined by scanning electron microscope (SEM).
The results showed that: (1) Numerous OCLs (TRAP-PMCs) were observed in an optimal coculture system established by OB (1x106 cells/6-cm dish) of new-born mice and BMCs (2x107 cells/6-cm dish) of adult female Wistar rats. However, the other five coculture models failed to develop TRAP-PMCs. Considering the uniformity of PEMFs, a 3.5-cm dish was substituted for the 6-cm dish and the optimal cell densities of OB and BMCs subsequently change into 4x105 and 2x107 respectively, and numerous TRAP-PMCs still appeared in this coculture system. (2) The results of apoptosis assay revealed that the rate of apoptotic OCLs increased when the apoptosis assay time increased, reaching the highest rate of 98%. Nuclear fragments of apoptotic OCLs appeared clearly after a longer time period after isolation. Compared with PEMF-1 and Control groups, the rates of apoptotic OCLs between 0~72 hr were similar. Compared with Control, the rate of apoptotic OCLs in PEMF-8 and PEMF-16 were both higher at 48 hr of apoptosis assay time points, and others were similar at 0, 24, and 72 hr of apoptosis assay time points.
It suggested that both PEMF stimulation time and apoptosis assay time points are important roles in inducing OCL apoptosis by PEMFs with specific parameters. The rate of apoptotic OCLs increased at 48 hr after exposure to PEMFs for 8 and 16 hr.

並列關鍵字

osteoblasts ； osteoclast-like cells ； bone marrow cells

參考文獻

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