利用電化學法偵測自然殺手細胞毒殺能力

A novel natural killer cell cytotoxicity assay by electrochemical approach

吳孟聰(Meng-Tsung Wu)

指導教授 ： 吳立真

國立暨南國際大學/科技學院/應用化學系/碩士(2010年)

https://doi.org/10.6837/NCNU.2010.00289

摘要

自然殺手細胞(NK cells)在免疫調節以及影響先天性與適應性免疫力上扮演重要的角色，牽涉到一些病毒和細胞內細菌感染的早期反應，且為對抗病毒感染的第一線。目前主要測量自然殺手細胞毒殺能力的方法，是將目標細胞 (target cells)攝入帶有放射性同位素的鉻離子(51Cr)與目標細胞表面抗原結合，當目標細胞被自然殺手細胞(NK cells)毒殺後，即可測量細胞所釋放出之鉻離子放射性強度，以推算細胞毒殺率。但此法牽涉到放射性物質之使用，造成實驗安全方面之顧慮。因此，本研究利用電化學偵測法，量測細胞所釋出之鉻離子，以期能替代放射性同位素的使用，同時又不失影響鉻離子偵測法在測量自然殺手細胞毒殺能力之穩定性與代表性。
本研究將4-吡啶硫醇（4-Mercaptopyridine, 4-MP）修飾於拋棄式金電極（三電極系統）表面，並與六價鉻離子產生錯合能力，在電極的偵測範圍內量測其還原電流，以推算出鉻離子的含量。此外，細胞攝入之鉻離子為六價，再經目標細胞代謝還原後轉為三價，此些三價鉻離子最後將釋放於細胞培養液中。因此，欲測量此些三價鉻離子含量必須先在鹼性條件下加入H2O2，以氧化成為六價鉻離子，其轉換率依10-4至10-6 M 的三價鉻依序為98、106及92.9 ％，之後再以電化學方波吸附剝除伏安法偵測出六價鉻的含量，進而推算出細胞毒殺率。
目前實驗結果為，在使用0.1 M H2SO4下，可測得之六價鉻離子線性範圍、靈敏度、偵測極限各為0.1 μM至10 μM 、0.764 μA / μM、 3.8 × 10-8 M。我們將再進一步利用電化學法所得到的偵測結果，與乳酸去氫酶分析法所得之細胞毒殺率做比較。

關鍵字

自然殺手細胞 ； 細胞毒殺率 ； 鉻離子 ； 方波伏安法

並列摘要

Radioactive materials have been widely applied in clinical diagnostics, agronomy, biology, cell biology, biochemistry, medical researches and environmental researches. One of the advantages of isotopic materials is the high sensitivity. However, the safety issue of radiation and the disposal of the radioactive wastes are the disadvantages of adopting this approach. In bio-related fields, various alternatives, such as colorimetric method and chemiluminescence have been developed to avoid the use of isotopic materials to protect operators from radiation. Many analytical approaches are involved in the use of 32P, 3H, 14C and 51Cr. In the field of immunology, 51Cr is used to measure the activities of CD8+ cytotoxic T lymphocyte (CTL) or natural kill cells termed chromium release assay. Target cells incorporated with 51Cr (VI) are recognized and lysed by activated lymphocytes. The measurement of the radioactivity (51Cr) released from target cells would be able to determine the abundance of activated lymphocytes. Herein, we designed a novel approach to analyze activated lymphocytes by using electrochemistry with square wave adsorptive stripping voltammetry (SWV) to measure chromium ion (Cr) instead of using 51Cr. After target cells were lysed, the released Cr was subjected to 4-mercaptopyridine (4-MP) surface modified disposable gold electrodes. The reduction current was enhanced due to the complexation between Cr (VI) and 4-MP. By analysis of the reduction current, the concentrations of Cr (VI) could be determined. Moreover, since target cells released Cr was in the Cr(III) state, it would be necessary to oxidized by H2O2 under basic condition before applying to the electrodes. Results indicated that the conversion rates of Cr (III) to Cr (VI) from 10-4M to 10-6M were 98, 106, and 92.9%, respectively. The medium concentrations of Cr (VI) could be referred to the activity of lymphocytes termed as cytotoxicity. The performance of the designed approach under 0.1M H2SO4 would be that the linear detection range: 10-7M~10-5M; the LOD: 3.8´10-8M; the sensitivity: 0.764 mA/mM.

並列關鍵字

natural killer cell ； cytotoxicity ； chromium ； SWV

參考文獻

1. 于善謙，王洪海，朱乃碩和葉容, 免疫學導論. 九州圖書文物有限公司, 2001.

2. Stites. Basic and clinical Immunology, 7th. 64.

3. Brittenden, J., et al., Natural killer cells and cancer. Cancer, 1996. 77(7): p. 1226-43.

4. Miles, M.P., et al., The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans. Acta Physiol Scand, 2002. 174(4): p. 317-25.

5. Perussia, B., et al., Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-alpha beta+, TCR-gamma delta+ T lymphocytes, and NK cells. J Immunol, 1992. 149(11): p. 3495-502.