細胞激素提升人類自然殺手細胞毒殺能力在細胞晶片上之分析與研究

Abstract

根據2008年國際癌症研究所(International Agency for Research on Cancer，IARC)的統計，美國血癌患者占所有癌症的9.9%，2010年台灣血癌死亡人數占所有癌症的4%。近年來已有不少運用自然殺手細胞成功治癒血癌的臨床案例，這種極具潛力的新興療法提供了治療血癌的新途徑。臨床研究結果顯示，自然殺手細胞毒殺活性與病患復發率有很大關聯性，如何篩選不同來源自然殺手細胞並在體外培養期間以細胞激素提升細胞活性與增殖數量，將是自然殺手細胞療法能否成功治癒血癌的關鍵。然而現有流式細胞儀毒殺率分析法需要很大數量的細胞(~10E6)，故一般不會實際用於臨床治療上。 本研究成功地應用細胞晶片偵測細胞激素活化之人類自然殺手細胞(NK92)對血癌細胞(K562)毒殺能力，僅需約90到150顆(10E1~10E2)人類自然殺手細胞與血癌細胞以效應細胞對目標細胞比例(Effector to target cell ratio，E:T) 2:1混和，於晶片中持續供應含有不同介白素配方、濃度的細胞培養液來活化人類自然殺手細胞，並透過即時光學觀測系統長時間觀察細胞反應與介白素活化之人類自然殺手細胞對血癌細胞毒殺率。實驗結果顯示細胞於晶片中培養18小時期間存活率在91%以上。結合介白素-2與介白素-12(IL-2+L-12)活化人類自然殺手細胞對血癌細胞之毒殺率明顯高過於單獨使用介白素-2活化，且兩者之細胞毒殺率差異將在6小時後逐漸變大。隨著介白素-12濃度增加自然殺手細胞對血癌細胞毒殺率會隨之提升，最經濟的介白素-12濃度為10 ng/ml，其18小時後細胞毒殺率平均為78.4%，此外細胞晶片亦可用於分析不同效應細胞對目標細胞比例下人類自然殺手細胞對血癌細胞之毒殺率。本研究之細胞晶片毒殺率分析結果與流式細胞儀分析結果相近，並具有實驗之可重現性。 相較於流式細胞儀毒殺率分析法，本研究之細胞晶片毒殺率分析法，其實驗設備與晶片製造成本低，僅需少量細胞樣本與試劑在晶片上長時間培養活化細胞同時分析細胞毒殺率，大幅降低實驗成本與實驗樣本試劑消耗量，更增加了實驗操作之便利性，可應用於篩選不同來源自然殺手細胞活性進而即時觀察細胞在體外培養期間受不同配方、濃度細胞激素活化的狀況，並選用細胞毒殺活性較佳的自然殺手細胞來治療血癌。

According to the statistical report reviewed by the International Agency for Research on Cancer, leukemia was responsible for 9.9% of all the numbers of cancer patients in the United States in year 2008. In recent years, quite a few clinical cases of people with leukemia been cured by natural killer (NK) cell transplantation had been observed. This therapeutic approach has the potential to be a promising new treatment for leukemia. Scientific investigations indicate a positive correlation in between the cytotoxic activity of NK cells and the remission in leukemia. In consequences, the selection of appropriate NK cells from donors or patients then activated by cytokines in vitro will be the key for the cure of leukemia. However, a great amount of NK cells (~106 cells) will be required in order to conduct such cytotoxic assay in flow cytometry, which may be regarded as impractical for the general clinic usages. This study has successfully demonstrated the application of the cell-based microdevice for the cytotoxic analysis of cytokines activated NK cells (NK92) against leukemic cells (K562). By using this biochip, it only requires around 90 to 150 (10E1~10E2) cells premixed at an E: T ratio of 2: 1 to conduct the cytotoxic assay. Also, the cytotoxic ability of NK cells can be activated by continuous supplying of culture medium with different concentrations or combinations of cytokines. Meanwhile, the long term cellular interaction and the cytotoxicity analysis of cytokines activated NK cells against K562 cells can be investigated via a real-time monitoring microscope system. The experimental results indicated that the viability of cells cultured on the biochip were more than 91%. The cytotoxicity of IL-2+IL-12 activated NK cells against K562 cells was significantly higher than the activation using IL-2 only. Furthermore the cytotoxicity difference increased with time after 6 hours. As the IL-12 concentration increased the cytotoxicity was enhanced accordingly. The most economical IL-12 concentration to activate NK cells was 10ng/ml, with the average cytotoxicity of 78.4%. Moreover, the cytotoxicity at different E: T ratios could be analyzed on the biochip. The cytotoxicity analyzed from the cell-based biochip was similar to the results in flow cytometry and the experimental data were reproducible. In contrasting with the flow cytometry assay, our cell-based microdevice requires small amount of cells (10E1~10E2) with minor quantity of experimental reagents to investigate long term cytotoxic ability of cytokines activated natural killer cells against leukemic cells. Through the use of cell-based microdevice combined with real-time microscope system, the experimental instruments and production costs are lowered. In sum, the microdevice in this study not only reduces the cost of cytotoxic assay but also increases the convenience of experimental manipulation. It can be apply in the NK cell therapy of leukemia for the activity selection of NK cells from different donors activated by different concentrations or combinations of cytokines.