肿瘤患者自体外周血来源CIK、NK细胞体外扩增影响因素及杀伤活性研究
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摘要
目的：探究肿瘤患者自身因素对CIK、NK细胞体外扩增能力的影响并检测其对肿瘤细胞的杀伤活性,为CIK、NK细胞在恶性肿瘤治疗中的应用进一步提供理论依据。
方法：第一部分：对既往于我科行CIK和NK自体细胞免疫治疗的恶性肿瘤患者进行回顾性分析,探讨患者年龄、性别、疾病种类、分期以及淋巴细胞亚群分布对CIK和NK细胞体外扩增能力的影响。在培养过程中每天计数细胞的数量,在培养前后分别应用倒置显微镜观察细胞的形态变化、应用流式细胞仪检测CD3+CD56+的CIK细胞以及CD3-CD56+NK细胞比例的变化情况。在培养的第14天收获细胞。第二部分：在培养的第7和第14天收获细胞,应用ELISA试剂盒检测CIK、NK细胞培养体系上清液中细胞因子IFN-γ的分泌水平。MTT法和Calcein-AM/PI荧光染色法检测CIK、NK细胞对K562细胞株的杀伤活性。
结果：1.肿瘤患者CIK、NK细胞的扩增能力低于正常人,但与年龄、性别、疾病种类无关。而疾病分期、淋巴细胞亚群分布等是影响CIK、NK细胞体外扩增能力的因素；Ⅲ-Ⅳ期患者CIK、NK细胞的扩增比例低于Ⅰ-Ⅱ期患者。而诱导后NK细胞的比例与诱导前NK细胞的比例呈正相关,与诱导前CD8+细胞的比例呈负相关。2.经过14 d的体外培养后CIK细胞的比例由2.03±0.17%升至30.78±15.29%,NK细胞的比例由11.32±3.42%升至58.32±10.13%,低于正常人外周血来源的CIK、NK细胞的扩增比例,细胞因子IFN-γ分泌水平分别升至10.0±0.44pg/mL、14.76±0.42pg/mL,均低于正常人CIK、NK细胞的分泌水平。3.诱导前肿瘤患者和正常人PBMC细胞的杀伤活性在各个效靶比时无差异,而诱导后CIK、NK细胞的杀伤活性较培养前PBMC细胞显著增强,随着效靶比的增加,CIK、NK细胞对K562细胞株的杀伤活性逐渐增强,尤以CIK联合NK细胞的杀伤活性最强,在效靶比为25：1时可高达80%。
结论：肿瘤患者CIK、NK细胞的体外扩增比例低于正常人,而与自身年龄、性别、疾病种类无关,但与疾病分期、淋巴细胞亚群分布相关。而诱导前患者外周血中NK细胞及CD8+细胞的比例与NK细胞的体外扩增能具有一定的相关性；可以通过体外诱导扩增获得大量的CIK、NK细胞,细胞因子IFN-γ的分泌水平及K562细胞株的杀伤活性显著增强,而CIK联合NK细胞的杀伤活性最强,可作为一种新型的过继免疫效应细胞应用于临床治疗恶性肿瘤。
Objective:To study the factors of cancer patients on the expansion ability of their own CIK and NK cells in vitro and detect the antitumor activity of the CIK and NK cells. In order to provide a theroretical basis for the treatment of CIK and NK cells against the malignant tumors.
Materials and methods:The first part, we analyzed the patiens with malignant tumors who had received CIK and NK autoimmune therapy, to explore the effects of age, gender, tumor type, stage and the distribution of the lymphocyte sub-group on the expansion of CIK and NK cells. we counted the number of the cells everday and observed the morphological changes before and after culture by inverted microscope, and the changes of CD3+CD56+ and CD3-CD56+ cells,and we harvest the CIK and NK cells at the 14th days. The second part, we detected the secretion level in the supernatant of the culture system by ELISA test kit. And we detect the killing activity of CIK and Nk against K562 cell line by MTT and Calcein-AM/PI fluorescence staining method.
Results:1. The expansion of the patients'CIK and NK cells was lower than the normal,but had no relevance with age, gender and the turmor type. However, the stage and the distribution of the lymphocyte sub-group were the factors in the expansion of CIK and NK cells. The expansion of the CIK and NK cells derived from stageⅢ-Ⅳpatients Was lower than the CIK and NK cells derived from stageⅠ-Ⅱpatients. After induction and expansion, the percentage of NK cells had a positive correlation with the percentage of NK cells before culture, and had a negtive correlation with the percentage of CD8+ cells before culture.2. After induction and expansion in vitro for 14 days,the percentage of CIK and NK cells raised to 30.78±15.29% and 58.32±10.13%, from 2.03±0.17% and 11.32±3.42%, respectively. The secretion level of IFN-γraised to 10.08±0.44pg/mL,14.76±0.42pg/mL,were lower than the normal CIK and NK cells.3. Before culture, the killing activity of PBMC from the patients and the normal was simililar at any effectors to targeted ratio. But after induction and expansion,the killing activity of CIK and NK cells against K562 cell line enhanced greatly compared with the PBMC. With the increase in effectors to targeted cells, the antitumor activity also increased at some degree. And the combining of CIK and NK cells had the most cytotoxicity,When the effectors to targeted cells was 25:1, the CIK and NK cells could kill the 80% targeted cells.
Conclusion:First, the expansion of the CIK and NK cells derived from cancer patients was lower than the normal. But had no relevance with the patients' age, gender and turmor type. But, the stage and the distribution of the lymphocyte sub-group were the factors in the expansion of CIK and NK cells in vitro. Second, we can obtain a large number of CIK and NK cells by induction and expansion in vitro, and the secretion level of IFN-γand the killing activity against K562 cell line were enhanced, and the combining of CIK and NK cells had the most cytotoxicity. Consequently, CIK and NK cells can be used to treat malignant patients in clinical as a new type of adoptive immuno effector cells.
方法：第一部分：对既往于我科行CIK和NK自体细胞免疫治疗的恶性肿瘤患者进行回顾性分析,探讨患者年龄、性别、疾病种类、分期以及淋巴细胞亚群分布对CIK和NK细胞体外扩增能力的影响。在培养过程中每天计数细胞的数量,在培养前后分别应用倒置显微镜观察细胞的形态变化、应用流式细胞仪检测CD3+CD56+的CIK细胞以及CD3-CD56+NK细胞比例的变化情况。在培养的第14天收获细胞。第二部分：在培养的第7和第14天收获细胞,应用ELISA试剂盒检测CIK、NK细胞培养体系上清液中细胞因子IFN-γ的分泌水平。MTT法和Calcein-AM/PI荧光染色法检测CIK、NK细胞对K562细胞株的杀伤活性。
结果：1.肿瘤患者CIK、NK细胞的扩增能力低于正常人,但与年龄、性别、疾病种类无关。而疾病分期、淋巴细胞亚群分布等是影响CIK、NK细胞体外扩增能力的因素；Ⅲ-Ⅳ期患者CIK、NK细胞的扩增比例低于Ⅰ-Ⅱ期患者。而诱导后NK细胞的比例与诱导前NK细胞的比例呈正相关,与诱导前CD8+细胞的比例呈负相关。2.经过14 d的体外培养后CIK细胞的比例由2.03±0.17%升至30.78±15.29%,NK细胞的比例由11.32±3.42%升至58.32±10.13%,低于正常人外周血来源的CIK、NK细胞的扩增比例,细胞因子IFN-γ分泌水平分别升至10.0±0.44pg/mL、14.76±0.42pg/mL,均低于正常人CIK、NK细胞的分泌水平。3.诱导前肿瘤患者和正常人PBMC细胞的杀伤活性在各个效靶比时无差异,而诱导后CIK、NK细胞的杀伤活性较培养前PBMC细胞显著增强,随着效靶比的增加,CIK、NK细胞对K562细胞株的杀伤活性逐渐增强,尤以CIK联合NK细胞的杀伤活性最强,在效靶比为25：1时可高达80%。
结论：肿瘤患者CIK、NK细胞的体外扩增比例低于正常人,而与自身年龄、性别、疾病种类无关,但与疾病分期、淋巴细胞亚群分布相关。而诱导前患者外周血中NK细胞及CD8+细胞的比例与NK细胞的体外扩增能具有一定的相关性；可以通过体外诱导扩增获得大量的CIK、NK细胞,细胞因子IFN-γ的分泌水平及K562细胞株的杀伤活性显著增强,而CIK联合NK细胞的杀伤活性最强,可作为一种新型的过继免疫效应细胞应用于临床治疗恶性肿瘤。
Objective:To study the factors of cancer patients on the expansion ability of their own CIK and NK cells in vitro and detect the antitumor activity of the CIK and NK cells. In order to provide a theroretical basis for the treatment of CIK and NK cells against the malignant tumors.
Materials and methods:The first part, we analyzed the patiens with malignant tumors who had received CIK and NK autoimmune therapy, to explore the effects of age, gender, tumor type, stage and the distribution of the lymphocyte sub-group on the expansion of CIK and NK cells. we counted the number of the cells everday and observed the morphological changes before and after culture by inverted microscope, and the changes of CD3+CD56+ and CD3-CD56+ cells,and we harvest the CIK and NK cells at the 14th days. The second part, we detected the secretion level in the supernatant of the culture system by ELISA test kit. And we detect the killing activity of CIK and Nk against K562 cell line by MTT and Calcein-AM/PI fluorescence staining method.
Results:1. The expansion of the patients'CIK and NK cells was lower than the normal,but had no relevance with age, gender and the turmor type. However, the stage and the distribution of the lymphocyte sub-group were the factors in the expansion of CIK and NK cells. The expansion of the CIK and NK cells derived from stageⅢ-Ⅳpatients Was lower than the CIK and NK cells derived from stageⅠ-Ⅱpatients. After induction and expansion, the percentage of NK cells had a positive correlation with the percentage of NK cells before culture, and had a negtive correlation with the percentage of CD8+ cells before culture.2. After induction and expansion in vitro for 14 days,the percentage of CIK and NK cells raised to 30.78±15.29% and 58.32±10.13%, from 2.03±0.17% and 11.32±3.42%, respectively. The secretion level of IFN-γraised to 10.08±0.44pg/mL,14.76±0.42pg/mL,were lower than the normal CIK and NK cells.3. Before culture, the killing activity of PBMC from the patients and the normal was simililar at any effectors to targeted ratio. But after induction and expansion,the killing activity of CIK and NK cells against K562 cell line enhanced greatly compared with the PBMC. With the increase in effectors to targeted cells, the antitumor activity also increased at some degree. And the combining of CIK and NK cells had the most cytotoxicity,When the effectors to targeted cells was 25:1, the CIK and NK cells could kill the 80% targeted cells.
Conclusion:First, the expansion of the CIK and NK cells derived from cancer patients was lower than the normal. But had no relevance with the patients' age, gender and turmor type. But, the stage and the distribution of the lymphocyte sub-group were the factors in the expansion of CIK and NK cells in vitro. Second, we can obtain a large number of CIK and NK cells by induction and expansion in vitro, and the secretion level of IFN-γand the killing activity against K562 cell line were enhanced, and the combining of CIK and NK cells had the most cytotoxicity. Consequently, CIK and NK cells can be used to treat malignant patients in clinical as a new type of adoptive immuno effector cells.
引文
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[2]Kalinski, P, Nakamura, Y, Watchmaker, P,et al. Helper roles of NK and CD8+ T cells in the induction of tumor immunity. Polarized dendritic cells as cancer vaccines [J]. Immunol,2006,36:137-146.
[3]尚敬婷.CIK细胞的基础和临床应用研究新进展[J].常州实用医学.2009,25：139-141.
[4]Verneris, MR, Baker, et al. Studies of ex vivo activated and expanded CD8+ NKT cells in humans and mice[J]. Clin. Immunol,2002,22:131-136
[5]Suradej Hongeng, Sawang Petvises, Surapon Worapongpaiboon,et al.Generation of CD3+CD56+Cytokine-Induced Killer Cells and Their In Vitro Cytotoxicity against Pediatric Cancer Cells[J].Hematology,2003, 77:175-179.
[6]HwanMookKim,JaeseungLim,Song-KyuPark,et al. Antitumor activity of cytokine-induced killer cells against human lung cancer[J]. International Immunopharmacology,2007,7:1802-1807.
[7]HwanMookKim,JaeseungLim,JongSoonKang,et al. Inhibition Of human cervical carcinoma growth by cytokine-induced killer cells in nude mouse xenograft model [J]. International Immunopharmacology,2009,9:375-380.
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