特定單脈衝電磁場對尼古丁所引發造骨細胞凋亡之抑制效果

Effects of Specific Single Pulsed Electromagnetic Field Stimulation on Inhibition of Osteoblast Apoptosis induced by Nicotine

許富發(Fu-Fa Hsu)

指導教授 ： 張恒雄 ； 詹文雄

中原大學/工學院/醫學工程研究所/碩士(2003年)

https://doi.org/10.6840/cycu200400654

摘要

尼古丁為香菸中最主要的成分，它是導致吸煙上癮的主因，而且會引起惡性腫瘤的產生。抽煙也是導致骨質疏鬆症的危險因子之一，同時尼古丁會對造骨細胞產生毒性反應，而本實驗室過去的研究發現特定參數之電磁場刺激能有效治療骨癒合，且促使造骨細胞增生，因此本研究之目的欲觀察電磁場對減緩造骨細胞因尼古丁而凋亡的效應，並於尼古丁刺激加單脈衝電磁場刺激後之不同時段分析造骨細胞凋亡比例，期許電磁場能達到減緩造骨細胞凋亡的作用，進而能防止骨質疏鬆症的產生。
本研究所使用造骨細胞細胞株為MC3T3-E1，初始培養密度為1×104/cm2，接受1μM~16mM之尼古丁濃度後，在4、8、12、16、24、48、72小時收集以MTT測定細胞活性，結果發現尼古丁會降低造骨細胞存活率，使造骨細胞死亡，此現象在高濃度尼古丁作用下更為明顯。接著進一步以西方點墨法來分析蛋白質，發現有PARP斷裂的情形，表示在尼古丁的作用下，造骨細胞會走向凋亡途徑。
而以磁場強度1.3 Gauss單脈衝電磁場刺激造骨細胞，在8、16與24小時後，能提升受4mM及8mM尼古丁刺激的細胞存活率，且此提升是藉由BCL-2蛋白質增加，而達到保護作用。
由本實驗可以知道磁場強度為1.3 Gauss之7.5 Hz單脈衝電磁場可以減緩造骨細胞的凋亡現象，增加造骨細胞存活率，對造骨細胞有一保護作用，維持造骨細胞數量。

關鍵字

細胞凋亡 ； 單脈衝電磁場 ； 造骨細胞 ； 尼古丁 ； BCL-2 ； poly ADP-ribose polymerase (PARP)

並列摘要

Smoking has been implicated as a risk factor in post- menopausal osteoporosis. The major component of the particulate phase of all cigarette smoke is nicotine. It could affect the regulation of bone mass. The balance between bone formation and resorption is determined by numbers and activity of osteoblast and osteoclast. Most prevalent metabolic bone diseases, such as osteoporosis, are due to an imbalance in bone remodeling. Single pulsed electromagnetic field (sPEMF) stimulations were confirmed to modulate the bone formation and resorption, which are carried out by osteoblasts and osteoclasts respectively.
This study demonstrated the effect of specific single pulsed electromagnetic field inhibiting osteoblast apoptosis induced by nicotine. Osteoblastic MC3T3-E1 cells were treated with nicotine (1μM to 16mM). At the same time, MC3T3-E1 cells were exposed by sPEMF (7.5Hz) at 1.3 Gauss for 8hr. There are two approachs used in this study to examine the apoptosis. The first approach is colorimetric tetrazolium (MTT) assay which is a method for determining the number of cell viability. MTT was converted into a purple formazan reaction product by redox of living cells. The formazan was then dissolved by DMSO and read on a microplate reader at a wavelength of 570nm. The second one is Western blot analysis method, which is an approach of analyzing proteins by probing immunoblots with antibodies against poly ADP-ribose polymerase (PARP) and Bcl-2.
Increasing of nicotine dose decreases the survival rate of MC3T3-E1 cells. In addition, the dose increase enhances the expression of PARP cleavage. It is known that the expression of PARP cleavage indicates the presence of damaged DNA. As a result cell apoptosis takes place. By knowing so, it is reasonable to assume PARP cleavage plays an important role in early apoptosis. These results suggest that nicotine be associated with the expression of cell death genes. In other words, nicotine can cause bone cells apoptosis.
We compared the survival rate of MC3T3-E1 cells between with and without sPEMF stimulations. The results of this study show the number of cells survived stimulated by sPEMF is significantly higher than those with sPEMF stimulation. This result is also verified by the Western blot assay method. In the Western blot analysis, we found the expression of Bcl-2 is enhanced. This finding indicates MC3T3-E1 cells are well protected and cell apoptosis is reduced.
Based upon the above study result, we conclude that nicotine can cause MC3T3-E1 cell apoptosis. Yet, higher dose nicotine aggravates cell apoptosis. Furthermore, sPEMF can reduce apoptosis of osteoblasts and increase the survival rate of osteoblasts by enchanced the expression of Bcl-2. This suggestes that sPEMF of 1.3 Gauss could reduce apoptosis of MC3T3-E1 cells and increase MC3T3-E1 cells viability.

並列關鍵字

apoptosis ； nicotine ； Bcl-2 ； poly ADP-ribose polymerase (PARP) ； Single pulsed electromagnetic field (sPEMF) ； osteoblast

參考文獻

蔡明慈，“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”，中原大學醫學工程研究所碩士論文，中壢，台灣，2002

Bassett CA, Pawluk RJ, Becker RO, “Effect of electrical current on bone in vivo,” Nature, Vol. 204, pp.652-655, 1964.

Bassett CA, Valdes MG. Hernandez E. “Modification of fracture repair with selected pulsing electromagnetic fields,” The Journal of Bone and Joint Surgery, Vol. 64, No. 6, pp.888-895, 1982.

Benowitz NL, “Interindividual variability in the metabolism and cardiovascular effect of nicotine in man.” Vol.221, pp.368-372, 1982

Benowitz NL. “The role of nicotine in smoking－related cardiovascular disease”. Pre Med, Vol.26, No.4, pp.412-417, 1997