跳到主要內容

臺灣博碩士論文加值系統

(44.222.64.76) 您好!臺灣時間:2024/06/14 07:48
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:許富發
研究生(外文):Fu-Fa Hsu
論文名稱: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
論文名稱(外文):Effects of Specific Single Pulsed Electromagnetic Field Stimulation on Inhibition of Osteoblast Apoptosis induced by Nicotine
指導教授:張恒雄詹文雄詹文雄引用關係
指導教授(外文):Walter H. ChangWalter H. Chang
學位類別:碩士
校院名稱:中原大學
系所名稱:醫學工程研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:97
中文關鍵詞:細胞凋亡單脈衝電磁場造骨細胞尼古丁BCL-2poly ADP-ribose polymerase (PARP)
外文關鍵詞:apoptosisnicotineBcl-2poly ADP-ribose polymerase (PARP)Single pulsed electromagnetic field (sPEMF)osteoblast
相關次數:
  • 被引用被引用:5
  • 點閱點閱:216
  • 評分評分:
  • 下載下載:18
  • 收藏至我的研究室書目清單書目收藏:0
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。
<a href="http://www.ntsearch.com/search.php?q=Smoking&v=56">Smoking</a> 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. <a href="http://www.ntsearch.com/search.php?q=Single&v=56">Single</a> 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 <a href="http://www.ntsearch.com/search.php?q=single&v=56">single</a> pulsed electromagnetic field inhibiting osteoblast apoptosis induced by nicotine. Osteoblastic MC3T3-E1 cells were treated with nicotine (1μM to 16mM). At the same <a href="http://www.ntsearch.com/search.php?q=time&v=56">time</a>, 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.
目 錄

摘要…………………………………………………………..…I
英文摘要………………………………………………………………III
誌謝……………………………………………………………………V
目錄……………………………………………………………………VI
圖索引…………………………………………………………………IΧ
表索引…………………………………………………………………ΧI

第一章 緒論………………………………………………………1
1-1 研究背景與目的……………………………………………1
1-2 尼古丁與骨癒合文獻回顧………………………………4
1-3電磁場文獻回顧……………………………………………5
1-4電磁場與抑制細胞凋亡文獻回顧………………………9

第二章 研究基礎………………………………………………10
2-1 尼古丁……………………………………………………10
2-2 骨骼………………………………………………………13
2-3 造骨細胞…………………………………………………13
2-4 骨細胞……………………………………………………15
2-5 蝕骨細胞…………………………………………………16
2-6 骨重塑作用………………………………………………18
2-7 細胞凋亡…………………………………………………21
2-8 細胞存活率測定…………………………………………28
2-9 鹼性磷酸?活性…………………………………………29
2-10脈衝式電磁場刺激系統…………………………………30
2-11 脈衝式電磁場刺激系統…………………………………29

第三章 材料與方法…………………………………………33
3-1 實驗設計…………………………………………………33
3-2 實驗材料…………………………………………………35
3-3 細胞培養模式……………………………………………38
3-4 鹼性磷酸?染色方法……………………………………40
3-5 細胞存活率測定…………………………………………42
3-6 蛋白質萃取與分析………………………………………43
3-7 西方點墨法步驟…………………………………………45
3-8統計分析…………………………………………………50

第四章 結果……………………………………………………51
4-1 造骨細胞特性鑑定結果…………………………51
4-2 尼古丁降低細胞存活率測定………………………54
4-3 有機溶劑對細胞存活率的影響………………………59
4-4 尼古丁對PRAP蛋白質的影響…………………61
4-5 電磁場對於抑制尼古丁降低存活率之結果…………62
4-6 電磁場對BCL-2蛋白質的影響…………………………65

第五章 討論……………………………………………………66
5-1 造骨細胞特性…………………………………………66
5-2 尼古丁影響造骨細胞……………………………………68
5-3 尼古丁作用於造骨細胞之凋亡分析……………………70
5-4 電磁場影響造骨細胞……………………………………71

第六章 總結……………………………………………………73
第七章 參考文獻………………………………………………75
附錄……………………………………………………………………82



圖 索 引

圖2-1 尼古丁結構式…………………………………………………10
圖2-2 骨重塑示意圖…………………………………………………20
圖 2-3 細胞壞死與細胞凋亡之型態區分………………………… 23
圖2-4 MTT機轉示意圖……………………………………………28
圖2-5 單脈衝電磁場原始輸出波形………………………………31
圖2-6 螺線圈與電磁場刺激器………………………………………32
圖2-7 電磁場刺激設備………………………………………………32
圖4-1 造骨細胞(MC3T3-E1) 造骨細胞之鹼性磷酸?染色………51
圖4-2 細胞培養20天Von Kossa染色測定骨小節生成…………52
圖4-3 細胞培養30天Von Kossa染色測定骨小節生成……………53
圖4-4 尼古丁作用24小時後造骨細胞株細胞存活率圖………… 55
圖4-5 尼古丁作用48小時後造骨細胞株細胞存活率圖…………55
圖4-6 尼古丁作用72小時後造骨細胞株之細胞存活率……………56
圖4-7 不同濃度尼古丁作用後造骨細胞存活率………………58
圖4-8不同濃度的溶劑,作用24小時對細胞存活率的影響………60
圖 4-9 尼古丁對造骨細胞的PARP影響……………………………61
圖 4-10電磁場與不同尼古丁濃度刺激之細胞存活率(8hr)………63
圖 4-11電磁場與不同尼古丁濃度刺激之細胞存活率(16hr)………64
圖 4-12電磁場與不同尼古丁濃度刺激之細胞存活率(24hr)………64
圖 4-13 尼古丁對造骨細胞的BCL-2影響………………………65



表 索 引

表2-1 細胞凋亡與細胞壞死比較表………………………………22
表3-1 標準曲線劑量…………………………………………………44
表 3-2 SDS-PAGE(1.5mm mini gel)配置…………………………49
表4-1 不同尼古丁濃度樣品中含乙醇的量…………………………59
附錄-1 尼古丁作用24小時,造骨細胞之細胞存活率……………82
附錄-2 尼古丁作用48小時,造骨細胞之細胞存活率…………82
附錄-3 尼古丁作用72小時,造骨細胞之細胞存活率…………82
附錄-4 尼古丁作用不同時間下,造骨細胞之細胞存活率…………83
附錄-5 尼古丁內含的溶劑,作用24小時對細胞存活率的影響……84
附錄-6 電磁場與不同尼古丁濃度刺激8小時之細胞存活率………84
附錄-7 電磁場與不同尼古丁濃度刺激16小時之細胞存活率……84
附錄-8 電磁場與不同尼古丁濃度刺激24小時之細胞存活率……85
Baron R, Chakraborty M, Chatterjee D, “Biology of the osteoclast. In Mundy GR, Martin TJ, Eds. Physiology and pharmacology of bone.” New York: Springer, pp.111-147, 1993

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, Hall S, Herning R, Peyton J, Jones R, Abdel-Latif O. “Smoker of low-yield cigarettes do not consume less nicotine.” N Eng J Med, Vol.309, pp.139-142, 1983

Benowitz NL “Pharmacologic aspects of cigarette smoking and nicotine addiction”, The New England J of Medicine, Vol.17, pp.1318-1330, 1988

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

Bilotta TW, Zati A, Gnudi S, Figus E, Giardino R, Fini M, Pratelli L, Mongiorgi R, “Electromagnetic fields in the treatment of postmenopausal osteoporosis: an experimental study conducted by densitometric, dry ash weight and metabolic analysis of bone tissue.” Chirurgia Degli Organi di Movimento, Vol. 79, No. 3, pp. 309-13, 1994

Broulik PD, “The effect of chronic nicotine administration on bone mineral content in mice.” Horm Metab Res, Vol.25, pp.219-221, 1993

Brighton CT, Luessenhop CP, Pollack SR, Steinberg DR, Petrik ME, Kaplan FS., “Treatment of castration-induced osteoporosis by a capacitively coupled electrical signal in rat vertebrae,” The Journal of Bone and Joint Surgery, Vol. 71, No. 2, pp.228-236, 1989.

Chang K, Chang WH, Wu ML, Shih C, “Effect of different intensities of extremely low frequency pulsed electromagnetic fields on formation of osteoclast-like cells.” Bioelectromagnetics, Vol.24, No.3, pp.189-98, 2003

Lohmann CH, Schwartz Z, Liu Y, Guerkov H, Dean DD, Simon B, Boyan BD, “Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production,” Journal of Orthopaedic Research, Vol. 18, No. 4, pp.637-646, 2000

Daniell HW “Postmenopausal tooth loss. Contributions to edentulism by osteoporosis and cigarette smoking.” Archives of Internal Medicine Vol.143, pp.1678-1682, 1983

Daftrari TK, Whitesides T, Heller J, Goodrich A, Mccarey B, Hutton W, “Nicotine on the revascularization of bone graft. An experiment study in rabbit.” Spine, Vol.19, pp.904-911, 1994

De Vernejoul MC, Bielakoff J, Herve M, “Evidence for defective by osteoblastic function. A role for alcohol and tobacco consumption in osteoporosis in middle-aged men.” Clinical Orthopaedics & Related Research , Vol.179, pp.107-115, 1983

Fang MA, Frost A, Iida-Klein A, Hahn T, “Effect of nicotine on cellular function in UMR-106 osteoblast-like cells.” Bone, Vol.12, pp.283-286, 1991


Griffiths RR, Henningfield JE, Bigelow GE. “Manipulation of number puffs perbout, interbout interval, and nicotine dose.” Journal of Pharmacology & Experimental Therapeutics, Vol.220, pp.256-265, 1982

Henningfield JE, Heishman SJ. “The addictive role of nicotine in tobacco use.” Phychopharmacology”, Vol.117, No.1, pp.11-13, 1995

Hernin RI, Jones RT, Benowitz NL, Mines AH. “How a cigarette is smoked determines blood nicotine level.” Clinical Pharmacology & Therapeutics, Vol.33, pp.84-90, 1983

Hoffman D, Adam J, “Carcinogenic tobacco-specific N-nitrosarmines in snuff and in the saliva of snuff dippers.” Cancer Research, Vol.41, pp.4306-4308, 1981

Rubin J, McLeod KJ, Titus L, Nanes MS, Catherwood BD, Rubin CT, “Formation of osteoclast-likecells is suppressed by low frequency, low intensity electricfields,” Journal of Orthopaedic Research, Vol. 14, No. 1, pp.7-15, 1996.

Heermeier K, Spanner M. Trager J, Gradinger R, Strauss PG, Kraus W, Schmidt J, “Effects of extremely low frequency electromagnetic field (EMF) on collagen type I mRNA expression and extracellular matrix synthesis of human osteoblastic cells,” Bioelectromagnetics, Vol. 19, pp.222-231, 1998.

Takayama K, Nomura H, Tanaka J, Zborowski M, Harasaki H, Jacobs GB, Malchesky PS, Licata AA, Nose Y, “Effect of a pulsing electromagnetic field on metabolically derived osteoporosis in rats: a pilot study,” Transactions – American Society for Artifical Internal Organs, Vol.36, No.3, pp.426-428, 1990.

Yonemori K, Matsunaga S, Ishidou Y, Maeda S, Yoshida H, “Early effects of electrical stimulation on osteogenesis,” Bone, Vol. 19, No. 2, pp.173-180, 1996.

Kelekar A, Thompson CB, “Bcl-2-family proteins: the role of the H3 domain in apoptosis”. Trends in Cell Biology, Vol. 8, No. 8, pp.324-30, 1998

Marks SC Jr, “Osteoblast biology: lessons from mammalian mutations.” American Journal of Medical Genetics, Vol. 34, pp.43-54, 1989

De Mattei M, Caruso A, Traina GC, Pezzetti F, Baroni T, Sollazzo V, “Correlation between pulsed electromagnetic fields exposure time and cell proliferation increase in human osteosarcoma cell lines and human normal osteoblast cells in vitro,” Bioelectromagnetics, Vol. 20, No. 3, pp.177-182, 1999.

Simko M, Kriehuber R, Weiss DG, Luben RA, “Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines,” Bioelectromagnetics, Vol. 19, pp.85-91, 1998.

Landry PS, Sadasivan KK, Marino AA, Albright JA, “Electromagnetic fields can affect osteogenesis by increasing the rate of differentiation,” Clinical Orthopaedics and Related Research, No. 338, pp.262-270, 1997.

Raikin SM, Landsman J, Alexander V, Forimson M, Plaxton N, Effect of nicotine on the rate and strength of long bone fracture healing.” Clinical Orthopaedics and related research, Vol. 353, pp.231-237, 1998

Ramp W, Lenz L, Galvin R, “Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in Osteoblast-like cells.” Proceedings of the Society for Experimental Biology & Medicine, Vol. 87, pp.36-43, 1991

Goodman R, Bassett CA, and Henderson AS, “Pulsing Electromagnetic Fields Induce Cellular Transcription,” Science, Vol. 220, No. 4603, pp.1283-1285, 1983

Riebel GD, Boden S, Whitesides T, Hutton W, “The effect o nicotine on incorpornof cancelloue bone graft in animal model.” Spine, Vol. 20, pp.2198-2202, 1995

Robison JG, Pendleton AR, Monson KO, Murray BK, O’Neill KL, “Decreased DNA repair rate and protection from heat induced apoptosis mediated by electromagnetic field exposure.” Bioelectromagnetics. Vol. 23, No. 2, pp.106-112, 2002

Rubin CT, McLeod KJ, Lanyon LE, “Prevention of osteoporosis by pulsed electromagnetic fields.” Journal of Bone and Joint Surgery, Vol. 71, No. 3, pp.411-7, 1989

Russell MA, Jarvis MJ, lyer R, Feyeraband C, “Relation of nicotine yield of cigarette to blood nicotine concentration of smoker.” British Medical Journal, Vol. 280, pp.972-976, 1980
Silcox HD, Daftrari T, Boden S, Schimandle J, Hutton W, Whitesides T, “The effect of nicotine on spinal fusion.” Spine, Vol. 20, pp.1954-1953, 1995

Mishima S, “The Effect of Long-Term Pulsing Electromagnetic Field Stimulation on Experimental Osteoporosis of Rats,” Journal of University of Occupational and Environmental, Vol. 10, No. 1, pp. 31-45, 1988

Stevenson JC, Lees B, Devenport M, Cust MP, Ganger KF, “Determinants of bone density in normal women: Risk factor of Osteoporosis”. Br Med J, Vol. 98, pp.924-928, 1989

Stolerman IP, Jarvis MJ. “The scientific case that nicotine is addictive. ” Psychopharmacology, Vol. 117, No. 1, pp.2-10, 1995

Suda T, Takahashi N, Martin TJ, “Modulation of osteoclast differentiation.” Endocrine Reviews, Vol. 13, No. 1, pp.66-80, 1992

Herkart PA. “Apoptosis: O death, where is the sting?.” J. Immunol, Vol. 154, No. 10, pp.4905-4908, 1995

Bodamyali T, Bhatt B, Hughes FJ, Winrow VR, Kanczler JM, Simon B, Abbott J, Blake DR, Stevens CR, “Pulsed electromagnetic fields stimultaneously induce osteogenesis and upregulate transcription of bone morphogenetic proteins 2 and 4 in rat osteoblasts in vitro,” Biochemical and Biophysical Research Communications, Vol. 250, No. 2, pp.458-461, 1998.

Tian F. Nakahara T. Yoshida. Honda N. Hirose H. Miyakoshi J. “Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells.” Biochemical & Biophysical Research Communications, Vol. 292, No. 2, pp.355-361, 2002

Tipton DA, Dabbous MK, “Effect of nicotine on proliferation and extracellular matrix production of gingival fibroblast in vitro. A preliminary retrospective study.” Journal of Periodontology, Vol. 22, pp.229-235, 1995

Skerry TM, Pead MJ, Lanyon LE, “Modulation of bone loss during disuse by pulsed electromagnetic fields,” Journal of Orthopaedic Research, Vol. 9, No. 4, pp.600-608, 1991.

Yamamoto TT, Kawakami M, Sakuda M, “Effect of a pulsing electromagnetic field on demineralized bone-matrix-induced bone formation in a bony defect in the premaxilla of rats,” Journal of Dental Research, Vol. 71, No. 12, pp.1920-1925, 1992.

Vermes I, Haanan C, “Apoptosis and programmed cell death in health and disease.” Advances in Clinical Chemistry, Vol.31, pp.177, 1994

Sollazzo V, Traina GC, DeMattei M, Pellati A, Pezzetti F, Caruso A, “Responses of human MG-63 osteosarcoma cell line and human osteoblast-like cells to pulsed electromagnetic fields,” Bioelectromagnetics, Vol. 14, pp.541-547, 1997.

Shankar VS, Simon BJ, Bax CM, Pazianas M, Moonga BS, Adebanjo OA, Zaidi M, “Effects of electromagnetic stimulation on the functional responsiveness of isolated rat osteoclasts,” Journal of Cellular Physiology, Vol. 176, pp.537-544, 1998.

Yuhara S, Kasagi S, Inoue A, Otsuka E, Hirose s, Hagiwara H, “Effect of nicotine on cultured cells suggest that it can influence the formation and resorption of bone.” European of Pharmacology, Vol. 383, pp.387-393, 1999

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

黃永彥,“骨質疏鬆症-基礎與臨床”,合記圖書出版社,台北,1997
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top