臺灣博碩士論文加值系統

睡眠呼吸中止症的病人，在睡眠中反覆地暫停吸入空氣，會導致間歇
性低氧，反覆地發生缺氧及重新獲得氧氣的循環相似於缺血再灌流傷害，
再獲得氧氣或再灌流會導致氧化壓力增加，在臨床上已知病人心血管疾病
危險性會增加，也會影響神經認知功能。
本實驗取 Sprague-Dawley rat 新生鼠的小腦組織，培養小腦顆粒細胞，
藉以探討間歇性低氧(20%氧氣和5%氧氣各30 分鐘交替，維持一天到四天)
是否會造成小腦顆粒細胞死亡增加，以及探討間歇性低氧造成小腦顆粒細
胞死亡的機制。
我們主要用共軛焦顯微鏡拍攝加上之後的電腦分析軟體的方式來獲
得實驗結果。首先，給予不同螢光染劑(HE，DCFDA)偵測細胞氧化壓力
(O2-•，H2O2，OH•)，當間歇性低氧時間逐漸增加時，細胞內的氧化壓
力就逐漸增加；再來利用Hoechst，PI 和TUNEL 等染色，偵測間歇性低氧
導致細胞死亡(包括細胞壞死和細胞凋亡)的比例，並給予不同的抑制藥物
阻斷引發細胞死亡的路徑，包括Fe chelator (phenanthrolin)以及PARP
inhibitors (3-AB，DPQ)，再觀察細胞死亡比例，當間歇性低氧時間逐漸增
加時，細胞死亡(包括細胞壞死和細胞凋亡)的比例增加，給予抑制劑後，
細胞死亡的比例就減少了；進一步探討導致細胞死亡的路徑，caspase 3 的
染色在間歇性低氧時間不同時並沒有變化，以西方點墨法偵測細胞內
caspase 3 也沒有看到活化的情形，而AIF 的染色發現，當給間歇性低氧時，
AIF translocation to nucleus 的比例增加，給予PARP inhibitor 3-AB 後比例
就減少。
因此，我們認為間歇性低氧會導致氧化壓力上升，導致細胞死亡增加，這
是因為PARP 活化，導致ATP 被消耗掉，以及AIF translocation to nucleus，
進而導致細胞死亡(包括細胞壞死和細胞凋亡)。

Episodic cessation of airflow during sleep in patients with sleep apnea
(SA) syndrome results in intermittent hypoxia. The condition with cycles of
hypoxia and reoxygenation resembles ischemia-reperfusion injury. Increased
oxidative stress caused by reoxygenation in SA patients has known to be
associated with increased risks of cardiovascular diseases and with
neurocognitive deficits.
The purposes of this study are to examine whether intermittent hypoxia
(20%O2 and 5%O2, 30minutes alternatively, 1 to 4 days) will induce increased
cell death of cerebellar granule cells and to find the mechanism of cell death
induced by intermittent hypoxia. Cerebellum from Sprague-Dawley neonatal
rats were removed for primary culture of cerebellar granule cells.
The results of our study were mainly from the pictures by confocal
microscope and then the pictures were analyzed with confocal software. At
first, cells were stained with HE and DCFDA to detect the cellular oxidative
stress (O2-•, H2O2, OH•). When the duration of intermittent hypoxia
increased, cellular oxidative stress elevated. Then cells were stained with
Hoechst, PI, and TUNEL to measure the percentages of cell death (apoptosis
and necrosis) induced by intermittent hypoxia. We gave several inhibitors to
block the cell death pathways, including Fe chelator (phenanthrolin) and
inhibitors of PARP (3-AB，DPQ), and measured the percentages of cell death
later. When the duration of intermittent hypoxia increased, the percentages of
cell death (apoptosis and necrosis) increased, and it decreased after inhibitors.
To examine the cell death pathways, cells were stained with caspase 3 and AIF.
The fluorescence of caspase 3 was not different in different duration of
intermittent hypoxia and caspase 3 activation was not detected by western blot.
II
However, we found that the ratio of AIF translocation to nucleus increased after intermittent hypoxia, and the ratio reduced after inhibitor of PARP (3-AB).
Therefore, intermittent hypoxia caused elevated oxidative stress, and then
induced cell death, which are mediated at least partly through activation of
PARP resulting in ATP depletion and AIF translocation to nucleus.

(3) AIF (apoptosis-inducing factor) － caspase independent
pathway……………………………………………………..7
間歇性低氧與小腦(Cerebellum)…………………………………..8
研究目的……………………………………………………………………..10
研究方法與材料……………………………………………………………..10
小腦神經顆粒細胞初級培養 (Primary cell culture)…………...10
間歇性低氧模式…………………………………………………...11
氧氣濃度偵測……………………………………………………...12
螢光染色偵測細胞氧化壓力…………………………………. …12
(1) 偵測O2
-•……………………………………………….…12
(2) 偵測H2O2 及OH•…………………………………….….12
螢光染色偵測細胞死亡…………………………………………...13
(1) 確認小腦顆粒細胞…………………………………….……13
(2) 偵測細胞凋亡(Apoptosis)…………………………….……13
(3) 偵測細胞壞死(Necrosis)……………………………………13
(4) Materials…………………………………………………….14
(5) 給予blockers 去抑制細胞死亡的步驟…………………….14
螢光染色偵測細胞內 caspase 3 及AIF…………………….……14
(1) 偵測caspase 3………………………………………….…..14
(2) 偵測AIF……………………………………………….…...14
共軛焦顯微鏡(Confocal microscopy)……………………………15
西方點墨法 (Western Blot)………………………………………15
(1) 全細胞蛋白質萃取(protein extraction)及蛋白質濃度測定
…………………………………………………………………...15
(2) 分離粒腺體及粒腺體的蛋白質萃取……………………….16
(3) 蛋白質濃度測定…………………………………………….16
(4) SDS-PAGE 及Transfer……………………………………..16
(5) 免疫染色……………………………………………………17
統計方法…………………………………………………………...18
研究結果……………………………………………………………………...19
間歇性低氧模式…………………………………………………...19
間歇性低氧造成氧化壓力上升……………………………...……19
間歇性低氧導致細胞死亡………………………………………...20
(1) 間歇性低氧導致細胞凋亡 (apoptosis)……………………20
(2) 間歇性低氧導致細胞壞死 (necrosis)……………………..20
間歇性低氧導致細胞死亡之機制探討…………………………...21
(1) 使用inhibitors 阻斷細胞死亡路徑………………………..21
(2) 偵測細胞內caspase 3 活化情形…………………………...21
(3) 偵測細胞內AIF 變化情形…………………………………22
(4) 西方點墨法 (Western Blot)……………………………….22
討論………………………………………………………………...…………24
本實驗的主要發現………………………………………………...24
間歇性低氧的傷害與 ischemia-reperfusion……………………..24
間歇性低氧導致氧化壓力上升及細胞死亡增加，兩者之間的關係
……………………………………………………………………...24
細胞死亡的路徑探討……………………………………………...25
PARP 活化與細胞壞死……………………………………………25
細胞凋亡與 caspase 3 活化之相關性…………………………….25
細胞凋亡與 PARP 活化和AIF 之相關性………………….……26
模式圖……………………………………………………………...27
顆粒細胞死亡與臨床影響………………………………………...27
實驗檢討…………………………………………………………...27
圖表與說明…………………………………………………………………...29
參考文獻……………………………………………………………………...51

Banasiak KJ, Haddad GG (1998) Hypoxia-induced apoptosis：effect of hypoxic
severity and role of p53 in neuronal cell death. Brain Res 797:295-304.
Barcelo A, Miralles C, Barbe F, Vila M, Pons S, Agusti AG (2000) Abnormal lipid
peroxidation in patients with sleep apnoea. Eur Respir J 16:644-647.
Barcelo A, Barbe F, Pena Mdl, Vila M, Perez G, Pierola J, Duran H, Agusti AG (2006)
Antioxidant status in patients with sleep apnoea and impact of continuous
positive airway pressure treatment Eur Respir J 27:756-760.
Bossenmeyer-Pourie C, Koziel V, Daval JL (1999) CPP32/caspase-3-like proteases in
hypoxia-induced apoptosis in developing brain neurons. brain Res Mol Brain
Res 71:225-237.
Cao G, Xing J, Xiao X, Liou A, Gao Y, Yin X, Clark R, Graham S, Chen J (2007)
Critical role of calpain I in mitochondrial release of apoptosis-inducing factor
in ischemic neuronal injury. J Neuroscience 27:9278-9293.
Carlson JT, Hedner J, Elam M, Ejnell H, Sellgren J, Wallin BG (1993) Augmented
resting sympathetic activity in awake patients with obstructive sleep apnea.
Chest 103:1763-1768.
Carpagnano GE, Kharitonov SA, Resta O, Foschino-Barbaro MP, Gramiccioni E,
Barnes PJ (2003) 8-Isoprostane, a marker of oxidative stress, is increased in
exhaled breath condensate of patients with obstructive sleep apnea after night
and is reduced by continuous positive airway pressure therapy. Chest
124:1386-1392.
Cherniack NS, Shenoy PC, Mishra R, Simonson M, Prabhakar NR (1996) Induction
of immediate early responses genes by hypoxia. Possible molecular bases for
systems a adaption to low pO2. Adv Exp Med Biol 410:127-134.
Chinopoulos C, Adam-Vizi V (2006) Calcium, mitochondria and oxidative stress in
neuronal pathology. FEBS 273:433-450.
Christou K, Moulas AN, Pastaka C, Gourgoulianis KI (2003a) Antioxidant capacity in
obstructive sleep apnea patients. Sleep Med 4:225-228.
Christou K, Markoulis N, Moulas AN, Pastaka C, Gourgoulianis KI (2003b) Reactive
oxygen metabolites (ROMs) as an index of oxidative stress in obstructive
sleep apnoea patients. Sleep Breath 7:105-110.
Dyken ME, Somers VK, Yamada T, Ren ZY, Zimmerman MB (1996) Investigating
the relationship between stroke and obstructive sleep apnea. Stroke
27:401-407.
Eric CCC, Lysanne M-D, Sean PC, Jacqueline LV, Kerry LF, William CM, David SP,
Steffany ALB, Ruth SS (2005) Apoptosis-inducing factor is a key factor in
neuronal cell death propagated by BAX-dependent and BAX-independent
52
mechanisms. Journal of Neuroscience 25:1324-1334.
Eric D, Santos A, Naoufal Z, Karine F, Theano I, Nathanael L, Marie C, Brian L,
David A, Josef P, Guido K (2000) Mitochondrio-nuclear translocation of AIF
in apoptosis and necrosis. FASEB J 14:729-739.
Fonfria E, Dare E, Benelli M, Sunol C, Ceccatelli S (2002) Translocation of
apoptosis-inducing factor in cerebellar granule cells exposed to neurotoxic
agents inducing oxidative stress. Eur J Neurosci 16:2013-2016.
Gozal D, Daniel JM, Dohanich GP (2001) Behavioral and anatomical correlates of
chronic episodic hypoxia during sleep in the rat. J Neuroscience
21:2442-2450.
Gozal E, Sachleben LR, Rane MJ, Vega C, Gozal D (2005a) Mild sustained and
intermittent hypoxia induce apoptosis in PC-12 cells via different mechanisms.
Am J Physiol Cell Physiol 288:C535-C542.
Gozal E, Shah ZA, Pequignot J-M, Pequignot J, Sachleben LR, Czyzyk-Krzeska MF,
Li RC, Guo S-Z, Gozal D (2005b) Tyrosine hydroxylase expression and
activity in the rat brain：differential regulation after long-term intermittent or
sustained hypoxia. J Appl Physiol 99:642-649.
Halestrap AP (2006) Calcium, mitochondria and reperfusion injury：a pore way to die.
Biochem Soc Trans 34:232-237.
Ip MS, Lam B, Lauder IJ, Tsang KW, Chung KF, Mok YW, Lam WK (2001) A
community study of sleep-disordered breathing in middle-aged Chinese men
in Hong Kong. Chest 119:62-69.
Kamata H, Hirata H (1999) Redox regulation of cellular signalling. Cell Signal
11:1-14.
Lavie L, Vishnevsky A, Lavie P (2004) Evidence fro lipid peroxidation in obstructive
sleep apnea. Sleep 27:123-128.
Lavie P, Herer P, Hoffstein V (2000) Obstructive sleep apnoea syndrome as a risk
factor for hypertension：population study. Br Med J 320:479-482.
Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431-1568.
Liu H, Colavitti R, Rovira II, T.Finkel (2005) Redox-dependent transcriptional
regulation. Circ Res 97:967-974.
Lossi L, Gambino G (2008) Apoptosis of the cerebellar neurons. Histol Histopathol
23:367-380.
Macey PM, Henderson LA, Macey KE, Alger JR, Frysinger RC (2002) Brain
morphology associated with obstructive sleep apnea. Am J Respir Crit Care
Med 166:1382-1387.
McCord JM (1985) Oxygen-derived free radicals in postischemic tissue injury. N
Engl J Med 312:159-163.
53
McCord JM (2000) The evolution of free radicals and oxidative stress. Am J Med
108:652-659.
Mehra R, Benjamin EJ, Shahar E, Gottlieb DJ, Nawabit R, Kirchner HL, Sahadevan J,
Redline S (2006) Association of nocturnal arrhythmias with sleep-disordered
breathing：the Sleep Heart Health Study. Am J Respir Crit Care Med
173:910-916.
Mishra RR, Adhikary G, Simonson MS, Cherniack NS, Prabhakar NR (1998) Role of
c-fos in hypoxia-induced AP-1 cis-element activity and tyrosine hydroxylase
gene expression. Brain Res Mol Brain Res 59:74-83.
Murad F (2004) Discovery of some of the biological effects of nitric oxide and its role
in cell signaling. Biosci Rep 24:452-474.
Naegele B, Pepin JL, Levy P, Bonnet C, Pellat J, Feuerstein C (1998) Cognitive
executive dysfunction in patients with obstructive sleep apnea syndrome
(OSAS) after CPAP treatment. Sleep 21:392-397.
Naegele B, Thouvard V, Pepin JL, Levy P, Bonnet C, Perret JE, Pellat J, Feuerstein C
(1995) Deficits of cognitive executive functions in patients with sleep apnea
syndrome. Sleep 18:43-52.
Narkiewicz K, Somers VK (2003) Sympathetic nerve activity in obstructive sleep
apnea. Acta Physiol Scand 177:385-390.
Nieto FJ, Young TB, Lind BK, Shahar E, Samet JM, Redline S, D'Agostino RB,
Newman AB, Lebowitz MD, Pickering TG (2000) Association of
sleep-disordered breathing, sleep apnea, and hypertension in a large
community-based study：Sleep Heart Health Study. JAMA 283:1829-1836.
O'Brien LM, Gozal D (2004) Neurocognitive dysfunction and sleep in children：from
human to rodent. Pediatr Clin North Am 51:187-202.
Pae E-K, Chien P, Harper RM (2005) intermittent hypoxia damages cerebellar cortex
and deep nuclei. Neuroscience Letters 375:123-128.
Peker Y, Kraiczi H, Hedner J, Loth S, Johansson A, Bende M (1999) An independent
association between obstructive sleep apnoea and coronary artery disease. Eur
Respir J 14:179-184.
Peppard PE, Young T, Palta M, Skatrud J (2000) Prospective study of the association
between sleep-disordered breathing and hypertension. N Engl J Med
342:1378-1384.
Sajkov D, Cowie RJ, Thornton AT, Espinoza HA, McEvoy RD (1994) Pulmonary
hypertension and hypoxemia in obstructive sleep apnea syndrome. Am J
Respir Crit Care Med 149:416-422.
Sauer H, Watenberg M, Hescheler J (2001) Reactive oxygen species as intracellular
messengers during cell growth and differentiation. Cell Physiol Biochem
54
11:173-186.
Schafer H, Koehler U, Ewig S, Hasper E, Tasci S, Luderitz B (1999) Obstructive
sleep apnea as a risk marker in coronary artery disease. Cardiology 92:79-84.
Shan X, Chi L, Ke Y, Luo C, Qian S, Gozal D, Liu R (2007) Manganese superoxide
dismutase protects mouse cortical neurons from chronic intermittent
hypoxia-mediated oxidative damage. Neurobiology of Disease 28:206-215.
Sheu S-S, Nauduni D, Anders MW (2006) Targeting antioxidants to mitochondria：A
new therapeutic direction. Biochimica et Biophysica Acta 1762:256-265.
Shimizu S, Eguchi Y, Kamiike W, Itoh Y, Hasegawa J, Yamabe K, Otsuki Y, Matsuda
H, Tsujimoto Y (1996) Induction of apoptosis as well as necrosis by hypoxia
and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. Cancer Res
56:2161-2166.
Somers VK, Dyken ME, Clary MP, Abboud FM (1995) Sympathetic neural
mechanisms in obstructive sleep apnea. J Clin Invest 96:1897-1904.
Tamatani M, Mitsuda N, Matsuzaki H, Okado H, Miyake S, Vitek MP, Yamaguchi A,
Tohyama M (2000) A pathway of neuronal apoptosis induced by
hypoxia/reoxygenation：role of nuclear factor-κβ and Bcl-2. J Neurochem
75:683-693.
Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol
552:335-344.
Wang Y, Dawson VL, Dawson TM (2009) Poly(ADP-ribose) signals to mitochondrial
AIF：A key event in parthanatos. Experimental Neurology.
Winterbourn CC (2002) Biological reactivity and biomarkers of the neutrophil
oxidant, hypochlorous acid. Toxicology 181:223-227.
Won SJ, Kim DY, Gwag BJ (2002) Cellular and molecular pathways of ischemic
neuronal death. J Biochem Mol biology 35:67-86.
Xu F, Frazier DT (2002) Role of the cerebellarr deep nuclei in respiratory modulation.
Cerebellum 1:35-40.
Xu W, Chi L, Row BW, Xu R, Ke Y, Xu B, Luo C, Kheirandish L, Gozal D, Liu R
(2004) Increased oxidative stress is associated with chronic intermittent
hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of
sleep apnea. Neuroscience 126:313-323.
Yaggi H, Mohsenin V (2004) Obstructive sleep apnoea and stroke. Lancet Neurol
3:333-342.
Yamauchi M, Kimura H (2008) Oxidant stress in obstructive sleep apnea：putative
pathways to the cardiovascular complications. Antioxid Redox Signal
10:755-768.
Yamauchi M, Nakano H, Maekawa J, Okamoto Y, Ohnishi Y, Suzuki T, Kimura