臺灣博碩士論文加值系統

PICK1 (Protein that interacts with C kinase 1) 是一個支架蛋白。實驗室過去研究在血癌細胞中位於粒線體上，並穩定膜電位，提高細胞對抗療藥物的抗性。而在小鼠纖維母細胞NIH3T3中，PICK1缺失的細胞其ATP產量降低、活性氧分子 (ROS) 含量上升且乳酸也較多，推測PICK1缺失影響粒線體功能。在低葡萄糖培養環境下會使其粒線體斷裂 (fission) 比例增加，並產生OPA1切割的現象；並引發自噬作用 (autophagy) 導致細胞死亡。本研究進一步探討在低濃度葡萄糖情況下補充葡萄糖 (glucose) 及麩醯胺酸 (glutamine) 對細胞死亡及OPA1切割的影響，我的結果顯示補充葡萄糖 (glucose) 能阻遏細胞死亡及OPA1的切割，顯示葡萄糖對於PICK1缺失細胞的存活是一個重要的因素。但在低濃度葡萄糖情況下外加麩醯胺酸 (glutamine) 卻無法阻遏細胞死亡及OPA1的切割，顯示麩醯胺酸雖然為主要細胞氮來源以及能量來源，但無法取代葡萄糖的必要性，而使細胞存活。另外，也發現在低濃度葡萄糖情況下，PICK1缺失的細胞株有AMPK蛋白表現較少的現象，而AMPK是一個調控能量代謝的蛋白，參與許多調控路徑，包含可能影響細胞自噬、能量代謝或粒線體更新等路徑。接著在葡萄糖缺乏的壓力下，以JC-1偵測粒線體膜電位的變化，實驗結果發現在低葡萄糖培養下，PICK1 缺失細胞其膜電位明顯低於NIH3T3，推測 PICK1蛋白的確對粒線體膜電位有其影響性，並推測於低葡萄糖培養下其能量供給本來就比正常狀態下不足，然而PICK1缺失細胞 (S8) 卻無法獲取更多能量，進而導致細胞死亡。

PICK1 (protein that interacts with C kinase 1) is a scaffold protein. Previous studies in this group have shown that PICK1 is localized to mitochondria, stabilizes the membrane potential and increases resistance to chemotherapeutic agents in leukemia cells. In NIH3T3 cells, we have shown that PICK1 deficiency caused a reduced ATP and an increased ROS and lactate. In addition, under a low glucose culturing condition, PICK1 deficiency significantly induced mitochondria fission, OPA1 processing and autophagic cell death. These results indicate an important role of PICK1 in mitochondrial functions. This study further investigated how supplements of glucose and glutamine might affect cell death and OPA1 processing. My results showed that supplement of glucose was sufficient to prevent low glucose-induced cell death and OPA1 processing, however glutamine, as a major nitrogen source and alternative energy source, was unable to rescue cell death and OPA1 processing. In addition, the levels of AMPK, an important protein involved in energy metabolism, were reduced in PICK1 deficient cells under low glucose culturing conditions as compared to NIH3T3 parental cells. I then investigated changes of mitochondrial membrane potential by JC-1 staining. My results showed that PICK1 deficient cells exhibited a significant lower mitochondrial membrane potential than NIH3T3 parental cells suggesting a role of PICK1 in maintaining mitochondrial membrane potential and PICK1 deficient cells failed to extract more energy under low glucose conditions which then led to cell death.

目錄 i
縮寫表 ii
圖次目錄 iv
附圖目錄 v
中文摘要 vi
英文摘要 vii
緒論 1
研究目標 12
實驗材料 13
實驗方法 17
結果 24
第一部分 探討不同養分對PICK1降低之NIH3T3細胞在葡萄糖缺乏環境下的影響 24
(一) 補充葡萄糖可以防止低葡萄糖誘導的細胞死亡現象 24
(二) PICK1降低會使得細胞在低濃度葡萄糖環境下引發OPA1切割
25
(三) 補充葡萄糖可以防止低葡萄糖誘導的OPA1切割現象 26
(四) 補充麩醯胺酸無法阻止低葡萄糖誘導的細胞死亡現象 26
(五) 補充麩醯胺酸無法阻止低葡萄糖誘導的OPA1切割現象 27
第二部分 探討PICK1降低之NIH3T3細胞在葡萄糖缺乏環境下對粒線體膜勢及能量代謝的影響 28
(一) PICK1降低之細胞AMPK蛋白表現量較NIH3T3少 29
(二) PICK1降低之細胞粒線體膜勢的變化 29
討論 32
參考文獻 37
圖次 46
附圖 61

圖次目錄
Figure 1. Knockdown of PICK1 promoted processing of OPA1 proteins under low glucose culture conditions. . 46
Figure 2. Glucose supplement was sufficient to prevent glucose deficiency-induced death. 48
Figure 3. Glucose supplement was sufficient to prevent glucose deficiency- induced OPA1 processing in PICK1-KD cells. 49
Figure 4. Glucose supplement at late stage was sufficient to prevent glucose deficiency- induced OPA1 processing in PICK1-KD cells. 50
Figure 5. Glutamine supplement could not prevent glucose deficiency- induced death. 52
Figure 6. Glutamine supplement could not prevent glucose deficiency- induced OPA1 processing in PICK1-KD cells. ... 54
Figure 7. High dose glutamine supplement could not to prevent glucose deficiency- induced death. 56
Figure 8. High dose glutamine supplement could not prevent glucose deficiency- induced OPA1 processing in PICK1-KD cells. 57
Figure 9. The protein levels of AMPK in PICK1 knockdown cells were reduced. ...... 58
Figure 10. Mitotracker Red fluorescence images of PICK1 knockdown cells. 59
Figure 11. The effect of low glucose condition on mitochondrial membrane potential of NIH3T3 and S8 cells. 60

附圖目錄
Figure S1. Aspects of glucose and glutamine metabolism. 61

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