恶性肿瘤患者外周血淋巴细胞高效体外扩增的方法

探讨从恶性肿瘤患者外周血单个核细胞（PBMCs）中高效扩增淋巴细胞的方法，取3例恶性肿瘤患者的PBMCs，每例患者重复3次，在GMP实验室体系下，经淋巴细胞刺激因子体外诱导扩增淋巴细胞和观察扩增情况，应用流式细胞仪分析扩增前后总CD3^＋、CD3^＋CD4^＋、CD3^＋CD8^＋淋巴细胞、CD3^＋CD16＋56^＋（NK细胞）、CD3^＋CD16＋56^＋（NKT细胞）以及CD4^＋和CD8^＋T细胞比值的变化，分析培养前后各淋巴细胞亚群的扩增情况和重复性，同时观察淋巴细胞体外扩增培养后回输患者的生物安全性。结果表明，CD3^＋、CD3^＋CD8^＋、CD3^＋CD16＋56^＋细胞比例较培养前明显增加（P〈0．01），而CD3^＋CD^＋、CD3^-CD16＋56^＋细胞比例、CD^＋和CD8^＋T细胞比值则明显的降低（P〈0．01）。CD3^＋、CD3^＋CD8^＋、CD3^＋CD16＋56^＋细胞增殖倍数的中位数分别为487．9、832．5和2540．6；而CD3^＋CD4^＋和CD3^＋CD16＋56^＋则分别为159．6和155．5。细胞体外扩增培养前后亚型和扩增倍数的重复性较差，在本研究中只有CD3^＋T细胞在体外培养前后、CD3^＋CD16＋56^＋细胞在培养后的CV值在10％以内。培养扩增后淋巴细胞细菌、真菌、支原体、外来病毒及内毒素检查后无阳性发现，所有患者细胞回输治疗后无明显的毒副反应。在本研究体系下体外诱导扩增培养肿瘤患者自体外周血NK细胞及致敏淋巴细胞效率较高，细胞毒性细胞比例增高较大，具有良好的生物安全性，为恶性肿瘤患者应用NK细胞进行过继免疫治疗提供了一种简单有效的扩增NK细胞的方法。

A Method of Expansion of Lymphocyte Cells from Malignant Neoplasm Patients' Peripheral Blood

To study the method of expansion of lymphocyte cells from malignant neoplasm patients＇ peripheral blood mononuclear cells （PBMCs） ,three neoplasm patients＇ PBMCs were cultured in the GMP laboratory system. Every patient was repeated three times. Inducing and expanding the lymphocyte cells and mononuclear cells by lymphocyte cell stimulating factors, observing the multiple of the expansion, to detect the percentage of CD3^＋、CD3^＋CD4^＋、CD3^＋CD8^＋, CD3^＋CD16＋56^＋, CD3^＋CD16＋56^＋ ceils in lymphocyte cells were detected by the flow cytometry, and then were compared their percentage and the rate of the CD3^＋CD4^＋ and CD3^＋CD8^＋ cells before and after the in vitro expansion. Furthermore, the repeatability of the expansion, and the biologic security of the lymphocyte cells after the in vitro expansion were observed and then transfusion to the patients. The result showed that after expanding, the percentage of CD3^＋ , CD3^＋ CD8^＋, CD3^＋CD16＋56^＋ cells was increased greatly （P 〈0.01 ）, and the percentage of CD3^＋ CD4^＋ , CD3^-CD16＋56^＋ cells and the rate of the CD3^＋CD^＋ and CD3^＋CD8^＋ cells were dropped obviously（P 〈0.01 ）. The median of expansion multiple of CD3^＋, CD3^＋ CD8^＋, CD3^＋CD16＋56^＋ cells were 487.9, 832.5 and 2540.6 separately, and the median of expansion multiple of CD3^＋CD4^＋ and CD3^-CD16＋56^＋ cells were 159.6 and 155.5. The repeatability of the expansion was not good, and only in the percentage of CD3^＋ cells before and after the expanding and the CD3^＋ CD16 ＋ 56^＋ cells after the expanding the values of CV was fewer than that 10%. There were no positive results on bacteria, epiphyte, mycoplasma, exogenous virus and endotoxin examination after in vitro expansion. There were no obvious adverse reactions on patients after transfusion. The in vitro induction and expansion efficiency of NK cells and sensitized lymphocyte cells from malignant tumor patients peripheral blood under this culture system is