探討脈衝式電磁場促進類骨母細胞增生之機轉

Study on the Mechanism of Osteoblast-like Cells Proliferation by Burst Pulsed Electromagnetic Field Stimulation

詹惠雯(Huei-Wen Jan)

指導教授 ： 張恆雄

中原大學/工學院/醫學工程研究所/碩士(2003年)

https://doi.org/10.6840/cycu200300593

摘要

本研究首先找出脈衝列電磁場（Burst pulsed electromagnetic field, bPEMF）促進類骨母細胞增生於體外培養下較佳的參數，再進一步探討在此參數下脈衝列電磁場誘發類骨母細胞增生的訊號傳導途徑。
實驗第一部份先架設適合體外培養模式使用之脈衝列電磁場刺激設備。以不同刺激時間的15 Hz脈衝列電磁場刺激不同細胞密度的類骨母細胞，並於最後一次刺激開始的24小時後，收取上清液，測定前列腺素E2（PGE2）濃度，並利用MTT分析法測定細胞生長力。第二部分則以第一部份實驗中最佳之脈衝電磁場刺激條件（磁場強度1Gauss、刺激時間8小時、骨母細胞分盤密度5 ×104 cells/cm2）刺激體外培養的類骨母細胞8小時。刺激開始的24小時後，收取上清液測定前列腺素E2（PGE2）濃度，並利用MTT分析法測定細胞生長力。實驗分成四組：控制組（不接受刺激）、試劑組（分別加入W-7、H-7、KT5720、KT5823、口引口朵 美哂辛（Indomethacin）或Bromophenacyl bromide（BPB）六種訊號傳導抑制劑，但不接受刺激）、刺激組，以及刺激加試劑組。
實驗結果發現，電磁場刺激能有效地促進細胞增生，且刺激組之PGE2濃度值與控制組比較均有顯著地下降趨勢（P＜0.05）。當在培養液中加入抑制劑時，抑制劑KT5720、KT5823以及BPB會抑制培養液內PGE2之濃度，可推測細胞合成PGE2之訊號途徑是經由活化PKA、PKG及PLA2的途徑來達成。另一方面，抑制劑H7、KT5823以及Indometnacin會抑制電磁場刺激細胞增生之作用，則可推測電磁場刺激是經由活化PKG途徑，最終達到引發細胞增生的作用。然而，電磁場刺激抑制細胞釋放PGE2的作用則會被抑制劑KT5823及BPB給抑制，因此推斷電磁場是藉由活化PKG途徑直接來引發細胞增生作用，並不是藉由先影響PGE2合成再進而造成細胞的增生。

關鍵字

Indomethacin ； 骨母細胞 ； W-7 ； 脈衝列電磁場 ； MTT分析 ； 前列腺素E2 ； 訊號傳導 ； KT5823 ； Bromophenacyl bromide ； H-7 ； KT5720

並列摘要

Burst pulsed electromagnetic flied (bPEMF) has been shown to have a significant impact in the healing of recalcitrant bone fractures in clinical trials and animal models. It has also demonstrated that bPEMF exposure accelerated bone defect healing and induced osteogenesis in vitro. However, the mechanism involved is still unclear. In this research, we studied the dosimetry effects of bPEMF on osteoblast-like cells proliferation in vitro, and investigated the biochemical pathways of that by some inhibitors.
We found that the optimum parameters of specific 15 Hz bPEMF were exposure time 8 hours (h), magnetic field 1 gauss, exposure duration 1 day with osteoblast seeding density of 5 × 104 cells/cm2. We exposed bPEMF on rat osteoblasts in the presence of various inhibitors of signal transduction, with or without bPEMF stimulation, and evaluated the stimulation effects on the cell growth by colorimetric tetrazolium (MTT) assay, and the secretion of prostaglandin E2 (PGE2) by enzyme-linked immunosorbent assay (ELISA). Six signal transduction inhibitors were used： W-7, a calmodulin antagonist; H-7, an inhibitor of cyclic nucleotide dependent protein kinase (PKA and PKG) and protein kinase C; KT5720, a specific, cell-permeable inhibitor of protein kinase A (PKA); KT5823, a selective inhibitor of protein kinase G (PKG); Indomethacin, which inhibits prostaglandin synthesis in the cell membrane; or Bromophenacyl bromide (BPB), which inhibits phospholipase A2 in the cell membrane.
bPEMF stimulations produced a significant increase in optical density (O.D.) of MTT assay and reduced PGE2 production compared with that in the control group. KT5720, KT5823 and BPB inhibitors prevented the PGE2 secretion of osteoblasts. It means that PGE2 production probably are mediated by PKA (block by KT5720), PKG (block by KT5823) and PLA2 (block by BPB) pathway activation. The stimulation effect of cell proliferation was inhibited significantly prevented increased cellular proliferation caused by stimulation. It means that PKG pathway (block by KT5823 and H7) probably are one of the important pathways of osteoblast proliferation promotion by PEMF exposure.

並列關鍵字

Bromophenacyl bromide ； Indomethacin ； Signal transduction ； KT5720 ； PGE2 ； MTT assay ； H-7 ； KT5823 ； Burst pulsed electromagnetic flied ； Osteoblasts ； W-7

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