Elucidation the mechanism of the immunological changes during anesthesia and abdominal surgery

本研究的主要目的之一是建立一种分析辅助性T细胞和自然杀伤细胞细胞因子发育规律的新方法。具有挑战性的新方法是：1)利用流式细胞术直接从单个核细胞亚群中提取辅助T淋巴细胞和自然杀伤细胞；2)用Elis POT法计算单个核细胞亚群中正在发育的细胞因子的细胞数；3)量化向Th1或Th2的分化和细胞因子的发育率的变化。1)用LAG-3和CD30双重染色的流式细胞术无法获得完全的重复性。2)用来评价细胞因子的变化，如TNFγ和IL-4的变化，不足以捕捉辅助性T淋巴细胞的微妙变化。3)我们正在发展一种新的方法，利用另一种标志物，如TNFβ、IL-5、IL-10、IL-13和CD4来评价辅助性T淋巴细胞的变化。该方法尚未建立，但我们很快就能成功地开发出该方法。4)为了评价作为反应细胞的Gammadelta-T细胞在热休克蛋白的发育过程中起重要作用并参与细胞凋亡的比率的变化，我们采用了CD3和TCR-γ/Delta双重染色的流式细胞术。5)认识上腹部手术期间外周血中Gammadelta-T细胞比率的变化。

One of the main purpose of this study was to develop the new method to analyze cytokine development pattern of helper T lymphocyte and natural killer cell. The challenging new method was following,1) Extract helper T lymphocyte and natural killer cell from subpopulations of mononuclear cell directly utilizing flow cytometry.2) Count the cell number of developing cytokine from subpopulations of mononuclear cell by ELIS POT method.3) Quantification of the differentiation to Th1 or Th2 and the change of the developmental ratio of the cytokines.The results were following,1 )We could not develop the methods to gain the complete reproducibility with flow cytometry utilizing double stain with LAG-3 and CD3O.2)The method to evaluate the change of cytokine development such as TNFgamma and lL-4 was not enough to catch the delicate change of helper T lymphocuytes.3)We are developing the new method to evaluate the change of helper T lymphocuytes utilizimg another markers such as TNFbeta, IL-5, IL-1O,IL-13 with cd4. The method has not be established yet but we will soon succeed in the develop of the method.4)To evaluate the change of the rates of gammadelta-T cell which is quite important as responding cell and participate to apotosis depended on the development of heat shock protein, we used the flow cytometry by the double stain with CD3 and TCR gamma/delta.5)The change of the rates of gammadelta-T cell in the peripheral blood during upper abdominal surgery was recognize.