鎘暴露對肝臟細胞的存活率與生物能量的影響及機制探討

Effects of cadmium exposure on hepatic viability and bioenergy

郭力瑋(Li-Wei Kuo)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2010年)

摘要

背景介紹：鎘，是一種存在我們日常生活環境中具有高毒性的一種重金屬。目前被廣為使用在許多工業中，例如：染劑、鎘鎳電池、塑膠的穩定劑，裡面都有鎘的存在。在人體中鎘會造成許多臟器的失能、特別是在肝臟。許多研究已經指出經過鎘的暴露過後會造成肝臟細胞的凋亡、壞死，而這些細胞的死亡與肝臟細胞粒線體的失能有密切關係，但是這其中的機轉還是尚須要繼續探討的。細胞內能量的多寡取決於ATP的製造與水解，大多數研究均指出在經過鎘的暴露後會造成粒線體電子傳遞鏈的破壞，以及細胞內ATP的下降。但對於ATP水解所佔的角色則較少被討論，因此本實驗將聚焦於鎘暴露後對於ATP水解的影響及IF1，一種調控ATP水解的蛋白質，在其中所扮演的角色。
材料與方法：我們利用clone 9這株肝細胞經過鎘處理後利用MTT assay進行細胞存活率、AproSENSOR kit偵測ATP含量、Flow cytometry測量粒線體膜電位、Western Blot偵測IF1蛋白質含量的估測以及Realtime PCR來測量IF1 mRNA的變化。
結果：經過了鎘的處理後，我們發現了細胞存活率在氯化鎘濃度10μM到100μM組呈現顯著的下降，而細胞內的ATP含量在氯化鎘濃度25μM、50μM組也有顯著的下降，IF1蛋白質的含量也在氯化鎘濃度12.5μM、25μM、50μM組也都有顯著的下降，IF1 mRNA的表現量在氯化鎘濃度25μM、50μM亦都呈現顯著的下降，粒線體的膜電位在氯化鎘25μM、50μM也有一個被破壞的情形，而粒線體水解ATP的活性在經過氯化鎘的處理過後也有一個顯著的上升。
結論：從以上結果我們推論，經過鎘的處理後IF1 mRNA的下降並伴隨著IF1蛋白質的被抑制及ATPase活性的上升，有可能是導致後續肝臟細胞內ATP含量的下降以及肝臟細胞死亡的一個重要原因。

關鍵字

鎘 ； 粒線體 ； 能量

並列摘要

Introduction：Cadmium is a highly toxic heavy metal in our environment. It is widely used in many industry such as：color pigment, batteries, plastic stabilizers. In human, cadmium causes organs dysfunction, especially in the liver. It has been reported that cadmium causes apoptosis, necrosis, which way be due to mitochondrial dysfunction in hepotocytes. However, the mechanisms are rare investigated. Several reports have revealed that mitochondrial respiratory enzyme activity decreases, leading to the decreased intracellular ATP content after cadmium exposure. Rare studies have been performed concerning about the hydrolysis effect of ATP after cadmium exposure. In this study, we focused on the in vitro effect of ATP hydrolysis after cadmium exposure, and the role of IF1, a regulator in ATP hydrolysis, is also investigated.
Materials and methods：We used clone 9 hepatocytes to observe the cell viability, ATP content, mitochondrial membrane potential, IF1 protein, the activity of ATP hydrolysis and the IF1 mRNA expression after cadmium treatment, and the mechanism was discussed.
Results：Our results showed that, after cadmium treatment, the cell viability, ATP content, IF1 protein level and the IF1 mRNA expression were significantly decreased simultaneously the mitochondrial membrane potential collapsed, and the activity of ATP hydrolysis was significantly increased in clone 9 hepatocytes.
Conclusion：We concluded that cadmium induces the decrease in cell viability of clone 9 cells, and the suppressed IF1 protein and the following increased ATP hydrolysis may play a role in bioenergetic failure after cadmium exposure.

並列關鍵字

cadmium ； mitochondria ； energy

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