利用共軛焦顯微鏡即時觀測與分析微流體晶片中細胞激素提升人類自然殺手細胞毒殺癌細胞之研究

血癌又稱為白血病，是指人體骨髓造血幹細胞（Hematopoietic stem cells,簡稱HSC）DNA變異產生癌化病變，不停地製造不正常之白血球，使得正常的白血球數量減少，不正常的白血球不斷的增生，於是將整個骨髓佔據並偏佈於血液中，已經嚴重影響正常血球的功能，導致身體很容易受到細菌感染。由於自然殺手細胞治療法副作用小，治療期間可維持病患相對較良好的生活品質，因此近年來有越來越多的患者選擇使用自然殺手細胞療法治療血癌。本研究利用微機電製程技術製作一微流體細胞晶片，藉由微流道設計與微流體操控技術，達到捕捉懸浮性細胞的功能。於細胞晶片中持續供應含有介白素配方之細胞培養液活化人類自然殺手細胞，並量測與分析細胞激素提升人類自然殺手細胞株(NK92-MI)對血癌細胞株(K562)之毒殺能力。以不同效應細胞對目標細胞比例(Effector to target cell ratio, E/T ratio) 混和及不同反應時間培養之樣品量測毒殺能力並分析其結果，利用流式細胞儀提供定量化資料，並與細胞晶片分析細胞激素活化之人類自然殺手細胞株(NK-92MI)對血癌細胞株(K562)毒殺能力之結果差異性作比較，以驗證細胞晶片分析毒殺能力之可行性。此外，藉由整合倒立式高解析度共軛焦顯微鏡，利用共軛焦顯微鏡掃描樣本不同的共軛焦面再由電腦重建影像的方式與螢光染色技術，可得到極高解析度與對比度之螢光影像，可做長期的追蹤觀測細胞間之反應行為，並將反應過程以影像紀錄，為自然殺手細胞毒殺癌細胞毒殺過程之定量化資訊。由結果顯示，K562被毒殺隨著E/T ratio的增加有提高之趨勢，亦即自然殺手細胞越多，則血癌細胞被毒殺的比例愈高。此外，於E/T ratio為2:1時，使用細胞晶片量測毒殺能力之結果與流式細胞儀所量測之結果最為相近。在分別將各組樣品以不同時間培養後量測毒殺能力，隨著2小時至18小時之量測結果，毒殺比率隨時間增加有提高趨勢，但趨勢漸緩，亦即毒殺能力有其限制。而於第18小時所量測毒殺率之結果顯示，加入介白素IL-12所激活之自然殺手細胞毒殺率相較於未使用者，其毒殺率從49.7%提升至79.2%，顯示藉由細胞激素IL-12激活自然殺手細胞效果顯著。將毒殺反應過程由共軛焦顯微鏡下觀察，K562細胞被NK92-MI細胞毒殺時，細胞形態上逐漸變化，包括:細胞萎縮、細胞膜出芽表現、及由凋亡細胞所產生的細胞凋亡體(apoptosis body)，其長期追蹤觀測細胞間反應行為之定性資訊可佐證定量之結果。

關鍵字

人類自然殺手細胞；微流體；生物晶片；微機電製程；共軛焦顯微鏡

並列摘要

Leukemia, known as blood cancer, occurs due to DNA variation of human bone marrow stem cells, making too many immature white blood cells. A large number of these immature white blood cells present in the blood seriously affect the normal function of blood cells. Due to little side effect of natural killer cell, natural killer cell therapy has been chosen as an auxiliary strategy in post chemical treatment for good quality of life. In this study, a microfluidic cell-trapped device was designed and manufactured by MEMS technique. With the design of narrow gaps between channels, suspension cells were trapped by a fluidic pressure drop between channels to be capable of trapped cell monitoring or observation under a microscopy. As a result, cell-cell interaction and cell apoptosis processes can be investigated with this device under a currently used fluorescence microscopy. In this study, cytokine interleukin 12 (IL 12) has been chosen to activate human natural killer cells in killing cancer cell (K562) process. With various ratios of effector cells to target cells (E/T ratio) in a cell-trapped device, a microfluidic cytotoxic assay was analyzed under a fluorescence microscopy. The cytotoxic results of the present device showed an excellent agreement with those by the conventional flow cytometry. The merit of the present device shows a feasible cytotoxic analysis using few cells in an order of 102 instead of around 106 cells for analysis in conventional flow cytometry. Additionally, the cytotoxicity of NK cells against cancer cells (K562) was found in a range from 49.7% to 79.2% over time periods in a cytokine IL-12 cell activation. Moreover, trapped in a device with an inverted high-resolution confocal microscopy by scanning fluorescent staining cell samples, the on-chip NK and cancer mixed cells in a cytokine-activated medium fluidics were kept monitoring in a long-term tracking of cell killing activities. Cell morphology was found to gradually change over time. Cell apoptosis process of cell shrinkage, membrane budding, and apoptosis bodies were firstly found with the present device in this experiment. Lastly, the on-chip determination of cell killing process and activity has been firstly achieved by this present device.

並列關鍵字

Natural killer cells ； MEMS ； microfluidics ； biochips ； confocal microscopy

參考文獻

[6]行政院衛生署中華民國99年癌症登記報告。

[2]D.A. Pollyea, H. E. Kohrt, and B. C. Medeiros, "Acute myeloid leukemia in the elderly: a review," Br J Haematol, vol. 152, pp. 524-42, 2011.

[3]C. Fausel, "Targeted chronic myeloid leukemia therapy: seeking a cure," American Journal of Health-System Pharmacy, vol. 64, pp. S9-S15, 2007.

[4]International Agency for Research on Cancer. Available: http://www-dep.iarc.fr/

[7]National Institutes of Health. Available: http://www.nih.gov/

國際替代計量

利用共軛焦顯微鏡即時觀測與分析微流體晶片中細胞激素提升人類自然殺手細胞毒殺癌細胞之研究

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