背景及研究目的：紅血球生成素（EPO）已被證明透過加強血管生成，以提高缺血器官的恢復。 EPO 的抗凋亡作用也在體外研究中被發現。為了理解潛在的機制和直接測試促紅血球生成素在肢體缺血急性期及之後血流恢復的抗凋亡的能力，在本研究於實驗過程，我們採用一個重症肢體缺血（CLI）的大鼠模型，來加以探討。
方法：測定JAK-2/STATs 訊號途徑在EPO 治療的重症肢體缺血增強恢復後的角色，雄性Sprgue-Dawley 大鼠（每組各12 隻），分為第一組（正常對照組），第二組（CLI 以生理鹽水治療），第三組（CLI 之後以促紅血球生成素治療），第四組（CLI之後以AG490，JAK-2 抑製劑治療），和第五組（CLI 之後以EPO 治療和AG490 處
理）。動物被犧牲的時間是在第1 天或第14 天，然後對缺血性股四頭肌做生化和病理組織學檢查 。
結果：在第1 天，給予EPO 的治療組顯示降低了凋亡的指數和活化了JAK2/STAT訊號途徑，其訊號途徑可透過額外AG490 的治療來抑制。此外，以EPO 治療和AG490處理，梗塞死亡面積減少，並且活化ERK1/2 和JNK，顯示了二者有類似的調節趨勢。值得注意的是，EPO 和AG490 對於增加抗氧化（GR，GPx，NQO-1）的表現方面
有協同作用並且也能減少發炎因子（TNF-α，NF-κB）的轉錄。在第14 天，單獨以EPO 或AG490 處理，亦或聯合治療，雷射都普勒分析顯示，皆可加強肢體缺血的血流恢復。
結論：雖然AG490 抑制JAK-2/STAT 途徑減少紅血球生成素在CLI 中早期階段有抗凋亡作用，但是重要的是AG490 的抗發炎和抗氧化是使CLI 之後能增強血流而扮演著正面的角色。

Background: Erythropoietin (EPO) has been demonstrated to enhance recovery in ischemic organ through enhancing angiogenesis. The anti-apoptosis action of EPO was also found in vitro study. To reveal underlying mechanisms and directly examine the benefits of anti-apoptotic capacity of EPO in acute phase of limb ischemia and following blood flow recovery, we applied an experimental critical limb ischemia (CLI) rat model in present study.
Methods: To determine the role of JAK2/STATs pathway in EPO-enhanced recovery after CLI, Male Sprague-Dawley rats (n=12 for each group) were divided into group 1 (normal control), group 2 (CLI treated with normal saline), group 3 (CLI treated with EPO), group 4 (CLI treated with AG490, a JAK2 inhibitor), and group 5
(CLI treated with EPO and AG490). Animals were sacrificed at either day 1 or day 14, followed by biochemical and histopathological examination on ischemic quadriceps.
Results: At day 1, EPO administration reduced expression levels of apoptotic indices and activated the JAK2/STAT pathway, which inhibited by additional AG490 treatment.Furthermore, the decrease of infarcted area, as well as activation of ERK1/2 and JNK,showed similar regulatory trends with EPO and AG490. Of Interest, EPO and AG490 showed a synergistic effect in increasing expression levels of antioxidants (GR, GPx,NQO-1) and in decreasing transcriptional levels of pro-inflammatory factors (TNF-α,NF-κB). At day 14, laser Doppler analysis showed that the blood flow recovery were enhanced with EPO, AG490, or combined treatment.
Conclusion: Although inhibition of JAK2/STAT pathways reduces the anti-apoptotic effects of EPO in early phase of CLI, the benefits of AG490 in anti-inflammation and anti-oxidation still play a positive role in enhancing blood flow recovery after CLI.

論文審定書………………………………………………………..............i
誌謝 ....…………………………………………………………...............ii
中文摘要 ..……………………………………………………............... iii
英文摘要…..……………………………………………………............. iv
目錄 ..…………………………………………………………..............vi
縮寫表 .……………………………………………………….............. viii
I. Introduction and Specific Aims …………………………............. 1
II. Materials and Methods……………………………………............ 5
III. Results …..………………………………………………............. 10
IV. Discussion ………………………………………………............. 17
V. Conclusion .………………………………………………............. 21
References ………………………………………………….............. 22
附圖
圖1: AG490 administration reduced the expression levels and activation of JAK2/STAT2, STAT5 which were increased by EPO treatment in ischemic limbs.....................…………………….... 28
圖2: EPO administration reduced the number of apoptotic nuclei and regulated mRNA expressions of apoptotic indices…………..................30
圖3: EPO reduced the infarcted area in ischemic quadriceps…....32
圖4: EPO and AG490 administrations reduced the mRNA expression levels of inflammatory factors and increased the expression levels of antioxidant …............................................. 33
圖5: The expression and phosphorylated levels of Akt, JNK, and ERK1/2 in ischemic quadriceps.....................................................34
圖6: EPO administration increased the expression levels of EPO receptors in ischemic limbs ..………………………………………36
圖7: EPO and AG490 administrations increased the blood flow recovery after critical limb ischemia induction in rats....…….......37
圖8: EPO and AG490 administrations increased the vessel density is quadriceps after critical limb ischemia induction in rats ...…....38

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