臺灣博碩士論文加值系統

Part-I
高鉀離子心臟麻醉灌流液(high KCl cardioplegia solution)是在心臟外科手術最常使用的方法。然而，高鉀離子心臟麻醉灌流液對心臟組織的副作用影響尚待釐清。目前的研究進一步探討鉀離子心臟麻醉灌流液是否誘發心肌細胞凋亡與心臟纖維化及其相關機轉。在冠狀動脈心臟病人進行高鉀離子心臟麻醉/冠動脈支路嫁接(CABG)治療之手術期間前後，游離小部分右心房組織(病人n = 20)。同時，使用鉀離子心臟麻醉灌流液及15 mM KCl solution處理H9c2心肌細胞、初代培養心肌細胞和初代培養纖維母細胞探討高鉀離子誘發心肌細胞凋亡與心臟纖維化的機轉。由TUNEL assay觀察到在CABG治療手術後的心臟組織檢體以及在高鉀離子溶液處裡過的心肌細胞都有明顯心肌細胞凋亡的現象。從Western Blot assay結果發現高鉀離子的確會誘發促凋亡蛋白（Bad, released cytochrome c and active caspase-3）大量表現。除此之外，高鉀離子更誘發non-cardiomyocyte ptoliferation-related IGF-I/IGF-IR/ERK pathway活化及cardiac fibrosis-related factors (uPA,
MMP-2, MMP-9, Sp-1 and CTGF)大量表現。然而心肌細胞survival
factor (Akt)表現量卻明顯地被高鉀離子抑制。綜合以上結果得知：高鉀離子透過誘發mitochondrial injury和Akt減少進而引起心肌細胞凋亡。同時伴隨著促非心肌細胞增生相關訊息途徑(IGF-I/IGF-IR/ERK pathway)活化與纖維化相關因子（MMP-2, MMP-9, Sp-1 and CTGF）的大量表現更加促使心臟功能衰竭的進行。此機轉解釋了心肌細胞凋亡如何在CABG治療之手術期間被誘發，並且暗示CABG治療手術之延長可能進一步引起心臟纖維化的癒後不全。

Part-II
先前我們的研究已發現，心室中隔缺損新生兒血清中的IGF-1濃度有
顯著性的下降，並且生長荷爾蒙會隨之增加。然而，為了確定IGF-1
及心室中隔缺損兩者之間的關係，我們進一步探討IGF-1及IGF-IR基
因在心室中隔缺損新生兒心肌組織的表現情形。首先，收集27位新生兒右心房組織進行研究。將組織進行分類: 5位沒有心室中隔缺損之新生兒分類為控制組(Group 1)；20位心室中隔缺損新生兒依據分流大小指數QP/QS(Shunting magnitude index)分成二組:中度分流(QP/QS <1.7)病患為實驗組二(Group II)，重度分流(Qp/Qs ≥ 2)患者為實驗組三(Group III)；此外，重度法洛氐四重症(TOF)以及分流指數Qp/Qs >4 新生兒則分類為實驗組四(Group Ⅳ)。利用非放射線DIG-RNA探針、西方墨點轉漬法及免疫組織化學染色等系統來測定心室中隔缺損新生兒右心房類胰島素生長因子及接受體基因表現。實驗結果顯示，在四個組別中，IGF-1以及IGF-IR蛋白質表現量分別為0.95 ± 0.05,100.42 ± 0.03, 0.29± 0.07, 0.23 ± 0.05以及0.79± 0.09, 0.58 ± 0.02, 0.37±0.02, 0.28 ± 0.04。另外IGF-1以及IGF-IR mRNA表現量為0.95 ± 0.01,0.41 ± 0.03, 0.29 ± 0.05, 0.15 ± 0.01及0.85 ± 0.05, 0.56 ± 0.03, 0.17 ±
0.01, 0.18 ± 0.01。與控制組(Group I)相比，Group II(p＜0.05)、GroupIII、Group Ⅳ(p＜0.01) IGF-1以及IGF-IR蛋白質及mRNA 表現量皆有顯著且逐漸減少的情形。在免疫組織化學染色結果也顯示相同的降低表現情形。此外，在心室中隔缺損的新生兒中，IGF-1、IGF-IR蛋白質及mRNA表現下降也與右心房飽和氧以及心臟收縮RVP/LVP比率有關。由以上實驗結果顯示，我們證實心室中隔缺損的新生兒心肌組織中IGF-1、IGF-IR表現量會下降，並且此現象與分流大小指數以及心臟腔室嚴重血氧缺乏有其關係性。

Part-I
Infusion of high KCl cardioplegia solution is the most common method for inducing asystole before cardiac surgery. However, the effects of cardioplegic solution on cardiac tissues and cells remain unclear. The present study examined the mechanisms behind cardiomyocyte apoptosis and cardiac fibrosis in patients who are administered high KCl cardioplegic solution prior to undergoing coronary artery bypass graft (CABG) to treat coronary artery disease (CAD). Two sequential biopsy
specimens were obtained from the right atriums during the
precardioplegic and postcardioplegic arrest periods in 20 CAD patients undergoing CABG surgery. In addition, cardiomyoblast H9c2 cells, primary neonatal cardiomyocytes and cardiac fibroblasts were treated with high KCl cardioplegic solution and 15 mM KCl solution to detect
the mechanisms behind cardiomyocyte apoptosis and cardiac fibrosis. TUNEL assay revealed increased levels of myocardiac cell apoptosis in biopsy specimens of the right atriums of patients who had been administered high KCl cardioplegic solution during CABG, and in H9c2 cells that had been exposed to high KCl cardioplegic solution and 15 mM KCl solution. Western blot showed increased activation of proapoptotic Bad, greater levels of released cytochrome c and active caspase-3 in postcardioplegic right atriums and in H9c2 cells exposed to high KCl cardioplegic solution and 15 mM KCl solution. The IGF-I/IGF-IR/ERK
pathway, known to be involved in cardiac fibroblast proliferation, and the expression/activation of uPA, MMP-2, MMP-9, Sp-1 and CTGF, which are known to be involved in the development of cardiac fibrosis, were up-regulated in postcardioplegic right atriums and cardiac fibroblasts.
However, the activation of survival factor Akt was greatly decreased in postcardioplegic right atriums and in cultured cardiomyocytes. These data suggest that high KCl cardioplegia appears to induce mitochondrial injury
and inactivation of Akt, resulting in apoptosis of cardiomyocytes. The activated IGF-I/IGF-IR/ERK pathway and the upregulation of uPA, MMP-2, MMP-9, Sp-1 and CTGF in cardiofibroblast cells further promote the development of cardiac fibrosis. The mechanism explains how cardiomyocyte apoptosis is induced during CABG surgery in CAD
patients, and might imply that the risk of cardiac fibrosis is dependent on the duration of surgery.

Part-II
Our previous studies showed serum insulin-like growth factor-I (IGF-I) concentrations significantly decreased in infants with congenital ventricular septal defect (VSD) and that they were associated with increased concentrations of growth hormone. In order to confirm the
relationship between IGF-I axis and VSD, we further compared the IGF-I and insulin-like growth factor-I receptor (IGF-IR) gene expressions in the cardiac tissue of VSD infants. Right atrium biopsies of 27 infants were
studied. Five infants not having VSD were classified as controls (Group I). Twenty VSD patients were then divided into 2 groups according to their shunting magnitude index (level of pulmonary vascular resistance compared with systemic vascular resistance, Qp/Qs). VSD patients with
minor shunts (Qp/Qs&lt;1.7) were classified as Group II; VSD patients with larger shunts (Qp/Qs&lt;2) as Group III. Besides, seven tetralogy of fallot (TOF) with shunt (Qp/Qs>4) infants were classified as the Group IV. A
nonradioactive DIG-RNA probe detection system, western blotting and immunohistochemistry were used to detect the gene expression levels and protein products of IGF-I and IGF-IR in the right atrium samples of VSD infants. The relative protein levels of IGF-I were 0.96±0.05, 0.43±0.03,
0.15±0.04, 0.12±0.03 and IGF-IR were 0.80±0.08, 0.57±0.03, 0.38±0.02, 0.24±0.04 in the right atrium of 4 group patients. The relative mRNA levels of IGF-I were 0.95±0.01, 0.41±0.03, 0.29±0.05, 0.15±0.01 and IGF-IR were 0.85±0.05, 0.56±0.03, 0.17±0.01, 0.18±0.01, respectively.
There was a significantly greater but more gradual decrease in protein levels and in mRNA levels of IGF-I and IGF-IR in Group II ( p&lt;0.05), Group III and IV (&lt;0.01) than in Group I. The results of immunohistochemistry also demonstrated a similar decrease in VSD patients. In addition, the decrease of mRNA and protein levels in IGF-I/
IGF-IR of VSD patients show related to the saturation of oxygen in the right atrium and the ratio of systolic right ventricular pressure to left ventricular pressure. We further confirmed the down regulation of IGF-I/IGF-IR in cardiac tissue of VSD infants and the decrease to be
associated with shunt magnitude and the severity of hypoxemia in the cardiac chamber of VSD.

目錄
壹、中文摘要 8
貳、英文摘要 11
參、前言與背景介紹 14
一、高鉀離子心臟麻醉劑 14
二、心肌細胞之計劃性凋亡 14
三、類胰島素生長因子 19
肆、研究動機 22
伍、研究材料與方法 23
一、新生兒心臟組織檢體收集 23
二、冠狀動脈繞道搭橋手術病患檢體收集 23
三、細胞培養 24
四、蛋白濃度測定 25
五、西方墨點法 25
六、siRNA 轉殖 26
七、MMP 活性分析 27
八、RNA 的萃取 27
九、Reverse Transcription/Polymerase Chain Reaction 28
十、IGF-I mRNA探針之製備 29
十一、Dot blotting 30
十二、組織切片 30
十三、TUNEL 細胞凋亡分析 31
十四、DAPI 細胞螢光染色分析 32
陸、實驗結果 34
高鉀離子心臟麻醉灌流液誘發心肌細胞凋亡及心臟纖維
化相關機制之探討
ㄧ、接受CABG 的CAD 病人右心房檢體具有大量
細胞凋亡之現象 34
二、高鉀離子心臟麻醉灌流液與KCl 溶液誘發H9c2
心肌細胞凋亡 34
三、高鉀離子心臟麻醉灌流液或KCl 溶液誘發CAD
病人右心房心肌細胞與H9c2 心肌細胞大量表
現促凋亡蛋白 34
四、接受CABG的CAD病人右心房檢體具有IGF-I
與IGF-IR表現量增加之現象 35
五、高鉀離子心臟麻醉灌流液和KCl溶液誘發H9c2
心肌細胞開啟IGF-I與IGF-IR基因並促進大量
表現IGF-I與IGF-IR蛋白 36
六、高鉀離子心臟麻醉灌流液開啟心臟纖維化訊息
途徑 36
七、高鉀離子心臟麻醉灌流液和KCl溶液誘發MMP-2
和MMP-9活性與表現量增加 37
八、MEK1 siRNA抑制高鉀離子心臟麻醉灌流液所
誘發uPA, MMP-2, MMP-9, Sp-1和CTGF的表現
量增加 38
類胰島素生長因子及其接受體基因在心室中隔缺損嬰兒
之心肌組織表現下降之探討
九、臨床個案之特徵及相關資料 39
十、利用Western blotting 比較各組間IGFI 與IGFIR
蛋白含量 40
十一、利用double-Y Plot software 比較各組間IGFI
與IGFIR 蛋白含量與病患病情嚴重的相互關
係 40
十二、利用dot blot 分析各組IGFI 與IGFIR 的mRNA
expression 41
十三、利用immunocytochemistry分析各組IGFI與IGFIR
的蛋白含量 42
柒、討論 43
捌、圖表說明 48
Figure 1. Cell apoptosis detection by TUNEL in right atrium
tissues of CABG patients and in high KCl-treated
H9c2 cells 50
Figure 2. Activation of Bad, caspase-3 and cytochrome c in
right atrium tissues of CABG patients and in high
KCl-treated H9c2 cells 52
Figure 3. Detection of IGF-1 and IGF-1R expression in right
atrium tissues of CABG patients 54
Figure 4. Detection of IGF-1 and IGF-1R expression in H9c2
cardiomyoblast cells treated with cardioplegic
solution and KCl solution 56
Figure 5. Detection of proteins related to cardiomyocyte
survival and cardiac fibrosis pathways in right
atrium tissues of CABG patients 59
Figure 6. Upregulation and/or activation of uPA, MMP-2,
MMP-9, Sp-1 and CTGF in postcardioplegic right
atriums, in H9c2 cells and neonatal cardiac fibroblasts
during cardioplegic solution treatment 61
Table 1. Characteristics of subjects in study 62
Figure 7. Western blotting analysis for insulin-like grow
factor-I, insulin-like growth factor-I receptor, and
α-tubulin in the right atrium of 4 group patients 63
Figure 8. Double-Y plot software to compare the tendency
of IGF-I or IGF-IR protein levels and RA SaO2,
RAP, RVP/LVP in each groups 64
Figure 9. Dot blotting analysis for IGF-I mRNA, IGF-IR
mRNA, and 18S rRNA in the right atrium of four
group patients 65
Figure 10. Immunocytochemistry of the right atrium in one
infant of each group 66
玖、參考文獻 67
拾、已發表之論文抽印本 75

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