人類自然殺手細胞對血癌細胞毒殺特性研究之生物晶片設計與開發

Abstract

血癌形成的原因是由於人體內免疫細胞病變，進而破壞人體的免疫系統，可經由骨隨移植的方式治療，不過捐贈者移植前需要和病患經過完整的人類白血球抗原(Human Leukocyte Antigen，HLA)配對，而只有約四分之一的病人可以在親屬間找到完全符合的HLA，病患經常花費大量時間尋找合適的捐贈者而延誤治療時間。自然殺手細胞屬於先天免疫(innate immunity)細胞，可分辨癌細胞並進行攻擊，最近臨床測試發現，捐贈者和病患的HLA不需要完全符合，移植治療後病患也不會產生排斥作用，雖然自然殺手細胞有上述優點，但不同捐贈者的自然殺手細胞特性都不相同，毒殺的能力也有差異，如何有效分析各種自然殺手細胞的毒殺活性，成為未來細胞治療很重要的依據。 本研究成功建立一生物懸浮細胞晶片，可捕捉固定數量之人類自然殺手細胞(NK92)以及血癌細胞(K562)形成不同的效能細胞對目標細胞比例(effector-to-target ratio)，先分別使用直徑10 μm、20 μm的微粒子作模擬，再以K562、NK92真實細胞進行捕捉數量測試，結果發現側邊縫隙長度和捕捉的細胞以及粒子數量成正比線性關係，與原先設計相符。由於，K562和直徑20 μm的微粒子大小尺寸相近，在縫隙長度相同條件下，兩者捕捉的數量相近；同樣地，NK92及10 μm微粒子也有相似的趨勢。側向縫隙長度180 μm以及720 μm分別平均捕捉10顆以及36顆K562細胞(樣本數=4)，而側向縫隙長度100 μm平均捕捉14顆NK92細胞(樣本數=4)。在細胞混合實驗中，本研究成功地在側向縫隙100 μm以及180 μm的微流道內捕捉17顆NK92以及10顆K562形成的效能細胞對目標細胞比例 17:10，並整合微氣動閥將細胞限制在特定區域中均勻混合接觸，結合量子點標定細胞技術，透過即時影像系統觀察NK92的毒殺特性。 不同於一般培養皿實驗混合大量細胞，觀察以群體的表現為主，本研究之微流道晶片，觀察少量細胞間作用行為，同時也節省了藥品的花費成本，可應用於觀測各式自然殺手細胞的活性。

Leukemia that occurs due to accumulation of abnormal white blood cells causes damage to the human immune system. A common treatment for Leukemia is used with bone marrow transplantation. Full Human Leukocyte Antigen(HLA) matched donor is required prior to bone marrow transplantation in order to prevent graft-versus-host disease (GvHD). The study reported that patients had only a limited chance of 25% finding the same HLA haplotypes from their own parents. Studies also have shown that natural killer cell transplantation with no need of HLA match is feasible for patients with leukemia. Natural killer cells are able to identify a wide range of tumor cells ,and to discriminate unhealthy cells efficiently. As a result, natural killer cell transplantation is considered as a promising treatment for leukemia. However, natural killer cells from healthy donors exhibit various levels of cytotoxicity to tumor cells. Effective investigation of cytotoxicity of natural killer cells to tumor cells remains a great challenge. We have developed cell-based micro-channel biosensors which are capable of forming various effector-to-target ratios, and of investigating the cytotoxicity of natural killer cells against leukemic cells. The cell-base biosensor with micro-channels was able to trap micro particles with 20 μm and 10 μm in diameter for simulation of leukemic cells (K562) and natural killer cells (NK92). It was found that the number of micro particles trapped in micro channel was proportional to the gap length. For real cells, the numbers of K562 trapped in micro channel with the gap length of 180 μm and 720 μm gap length were 10 and 36, respectively (sample=4). In the case of a gap length of 100 μm, the device was found to trap 14 natural killer cells in average(sample=4). By use of micro-particle simulation, this study shows similar tendency of real cells of tumor cells (K562) and natural killer cells (NK92) in this device. The cell-based biosensor device achieved the effector (NK92) - to - target (K562) ratio of 17 /10 in this study, and allowed the real-time monitoring microscopic system for cytotoxic process and effective investigation. Unlike conventional cell culture experiments in a dish of a huge amount of cells, the present device simply allows very few hybrid cells for manipulation, cytotoxic interaction, and significantly reduced consumption of culture medium. The cell-based micro device exhibits the capability and potential of effective investigation for tumor cells (K562) and natural killer cells(NK92).