利用非放射性鉻離子偵測方法測量毒殺型T淋巴細胞毒殺率

細胞型免疫在後天免疫系統中扮演很重要的角色，主要是由兩種T淋巴細胞進行，分別為輔助型T淋巴細胞和毒殺型T 淋巴細胞；當抗原呈獻細胞呈獻抗原給毒殺型T 淋巴細胞時，此時毒殺型T 淋巴細胞即被活化並大量的增生以消滅被感染的細胞。目前主要測量毒殺型T淋巴細胞毒殺率的方法，是將帶有放射性同位素的鉻離子(51Cr)與目標細胞表面抗原結合，當目標細胞被毒殺型T 淋巴細胞毒殺後，鉻離子即被釋放，因此利用測量放射性強度來推算細胞毒殺率。 Luminol為化學發光反應中常用的發光試劑，Luminol與雙氧水加上適當的催化劑，即會產生化學冷光，而鉻離子(III)即是催化此反應的催化劑之一。我們在細胞培養液中加入鉻離子(VI)，鉻離子(VI)會經由細胞膜上的離子通道進入細胞內，在細胞內由六價還原成三價，三價鉻離子則留在細胞內無法到細胞外，因此當毒殺型T 淋巴細胞將細胞殺死細胞之後，細胞內的鉻離子即被釋放到溶液中，因此我們利用毛細管電泳配合光電倍增管作為化學冷光偵測器，藉由偵測化學冷光推算溶液內的鉻離子濃度，進而推算出細胞毒殺率；細胞毒殺率再與乳酸去氫酶分析法(LDH assay)所測量的結果比較。本論文中，我們嘗試比較不同緩衝溶液、不同發光試劑濃度等條件下，來找出最佳條件。結果在0.02 M，pH 4.7的醋酸緩衝溶液，Luminol濃度10 mM，雙氧水濃度10 mM的條件下，達到最佳偵測極限。此外，利用乳酸去氫酶分析法(Lactate dehydrogenase assay, LDH assay)已成功進行細胞毒殺試驗。進一步將再利用化學冷光偵測系統，來與乳酸去氫酶分析法所得到的細胞毒殺結果比較。

關鍵字

毒殺型T淋巴細胞；細胞毒殺率；化學冷光；毛細管電泳

並列摘要

Cell-mediated immunity is one of the key elements of the adaptive immune responses. This type of immunity is mediated by T lymphocytes such as CD4+ helper T lymphocytes and CD8+ cytotoxic T lymphocytes (CTL). The stages of cell-mediated immunity consist of the activation and proliferation of naïve T cells to become effector cells and the elimination of infected cells. The interaction of CD8+ T cells with epitopes displayed by MHC molecules on antigen presenting cells ensures the specificity of T cells on infected cells. The determination of activated CD8+ cytotoxic T cells (CTL) is therefore important for the assessment of cell-mediated immunity. The classical assay for CTL activity is the chromium release assay. Target cells expressing antigen on their surface are labeled with a radioactive isotope of chromium (51Cr). Soluble Cr (VI) traverses the cell membrane by non-specific anion transporters followed by intracellular reduction to chromium (III), which is unable to cross the membrane. The antigen-specific lysis is calculated by the measurement of the released 51Cr in the medium after the attacking of specific CD8+ T cells toward their target cells. Various methods for the determination of CTL have been developed. 51Cr-release assays, however, is the most reliable and sensitive. One of the disadvantage of 51Cr-release assays approach is the use of radioisotopes. Therefore, the development of an easy-to-use, speed, sensitive, high accuracy and safety approach is in an urgent. In this study, a non-radioactive chromium release assay with the aid of capillary electrophoresis and chemiluminescence detection is developed. Chromium (III) catalyzes the reaction of luminol and hydrogen peroxide in basic condition to generate chemiluminescence, which can then be detected by photomultiplier tube (PMT) system. The factors that influence the intensity of the signals include the concentrations of luminol, hydrogen peroxide and chromium (III) ions. This system is fast and safe for determination of CTL activity.