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研究生:葉怡亨
研究生(外文):I-Jeng Yeh
論文名稱:探討N-acetylcysteine治療鉻引發腎細胞傷害之最佳劑量與機轉
論文名稱(外文):Optimal regimen and mechanisms of N-acetylcysteine on chromium-induced renal cells damage
指導教授:彭瓊瑜彭瓊瑜引用關係
指導教授(外文):Chiung-Yu Peng
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:公共衛生學系職業安全衛生碩士班
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:59
中文關鍵詞:細胞凋亡六價鉻人體近端腎小管細胞乙醯半胱氨酸活性氧類
外文關鍵詞:ApotosisHexavalent chromiumHuman proximal tubular epithelial cellN-acetylcysteineReactive oxygen species
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鉻對人體是有害之重金屬,特別對腎臟與肺臟最常導致傷害及死亡。鉻廣泛使用於工業及農業中,職業暴露與中毒是無可避免的。然而目前臨床上仍缺乏經證實有效之治療策略,因此發展對鉻有效之治療方法是有其急迫性與必要性。目前已知鉻中毒機轉是經由細胞內產生的氧化物,造成細胞功能異常、自毀與死亡。因此抗氧化物如NAC可能扮演重要之解毒角色。雖然臨床上已使用NAC做為鉻中毒的解毒劑,但其效果仍無足夠證據支持,此外NAC在治療鉻中毒之最佳劑量、給藥時間與治療機轉仍未確定。
藉由MTT方法評估人類近端腎小管細胞 (HK-2 cells)在鉻暴露後之存活比率,測試在不同時間,加入不同劑量之NAC後,細胞存活比率是否有顯著差異,藉而找出最佳劑量與給藥時間。另外藉由流式細胞儀(flow cytometry)偵測細胞凋亡與自由基的產生,了解自由基與細胞凋亡的時序相關性;此外,利用蛋白質轉漬法(Western Blot)分析PARP、caspase-3、caspase-8、caspase-9、cytochrome C、Bax、Bcl-xL、death receptor complex等等的改變,來了解NAC如何能阻斷鉻所造成之細胞傷害。
本研究結果顯示HK2細胞在六價鉻的傷害後,如能在2個小時內給予達600 μg/mL濃度的NAC時,細胞的存活率有顯著增加。同時在自由基的研究,NAC展現降低自由基產生的效果。針對HK2細胞凋亡,本研究發現鉻會引發內外路徑的同時活化。而且在MTT研究中,最佳NAC策略也是唯一能同時抑制內外路徑的方式。
根據以上結果,本研究認為最佳NAC的劑量必需達到600μg/mL的濃度,且其最佳治療時間為中毒後兩小時內。針對內外途徑的抑制,會因NAC的治療時間而呈現不同效果。


Chromium is a well-known heavy metal that mainly causes renal and lung damages. Due to increasing industrial and agriculture application, the risk of occupational exposure and poisoning are inevitable. Currently, the lack of valid effective treatment strategies would emerge the need to develop an optimal therapeutic modality. The production of reactive oxygen species (ROS) by chromium intoxication is the main mechanism to induces cell dysfunction, apoptosis and death. A potent antioxidant medication, such as N-acetylcysteine (NAC), might be a preferable antidote. Although NAC had been used as chromium intoxication antidote, there is no strong evidence to support its therapeutic efficacy. Moreover, its optimal dose and tolerable time gap of delayed treatment have not been clarified yet.
By MTT assay, the viability of HK-2 cells after chromium exposure was evaluated. The effect of NAC of various concentrations on cell viability after chromium exposure was tested at different time periods. Flow cytometry was used to detect the status of cell''s death and the generation of free radical. Additionally, we used western blot to analyze the expression of PARP, caspase-3, caspase-8, caspase-9, cytochrome C, Bcl-xL, Bax, and death receptor complex that may help us to understand the mechanism of NAC to block chromium-induced cell damage.
Our results showed cell viability clearly increased after the supplement of NAC to block chromium-induced cell death, particularly when 600 μg/mL NAC was given within 2 hours after chromium exposure. Moreover, free radical generation was also blocked by the supplement of NAC. In apoptotic signaling, chromium induced apoptosis by activation of both intrinsic and extrinsic pathways. Therefore, the NAC regimen might be the best way that is capable to inhibit both apoptotic pathways.
In conclusion, the optimal dose and treating-time point of NAC was 600 μg/mL supplemented and within 2 hours of chromium intoxication. Moreover, the efficacy of NAC to inhibit both apoptosis pathways was variable according to the time of its therapeutic application.


Table of Contents
Chapter 1. Introduction P.09
Description P.09
Acute human toxicity P.10
Mechanism of toxicity P.10
Apoptosis P.11
Treatment strategies P.14
N-acetylcysteine P.15
Chapter 2. Objective P.18
Chapter 3. Materials and Methods P.20
HK-2 cell culture P.20
MTT assay for cell viability P.20
Oxidative Stress Assays P.21
Apoptotic assay by Annexin V staining P.22
Apoptotic assay by Western Blot analysis P.23
Statistical Analysis P.25
Chapter 4. Results P.26
Cytotoxic effect of Potassium dichromate and N-acetylcysteine P.26
Early NAC rescue was needed to limit chromium-induced renal damage P.28
Anti-oxidative NAC decrease chromium-induced oxidative stress in HK-2 cells P.30 
Chromium induced HK-2 Cell death via apoptotic pathway P.31
NAC inhibited chromium-induced apoptosis through intrinsic and extrinsic apoptotic pathways was dependent on supplement time P.34
The appearance of cytochrome C in cytoplasm was not associated with it decrease in mitochondria P.38
FasL-soluble form might have active role in the activation of extrinsic apoptotic pathway during chromium intoxication P.40
Chapter 5. Discussion P.42
Chapter 6. Conclusion P.51
References P.52


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