IL-6、PHA联合磁珠分选对CIK增殖能力和杀伤肾癌786-O细胞功能的影响

The effect of IL-6、PHA and IMB sorting on proliferation and function of CIK cells

目的通过体外实验研究白介素-6(IL-6)、植物血凝素(PHA)联合免疫磁珠(IMB)分选对CIK增殖能力和杀伤肾癌786-O细胞功能的影响,为肾脏肿瘤的免疫治疗提供理论依据。方法分离肿瘤患者外周血单个核细胞(PBMC),经Mini MACS免疫磁珠去除CD4+CD25+Treg细胞后分为4组:对照组以CD3 Mc Ab、IL-2和IFN-γ诱导培养;PHA-CIK组、IL-6-CIK分别在对照组的基础上加用PHA、IL-6;PHA-IL-6-CIK组在对照组的基础上加用PHA和IL-6。CCK-8法检测各组CIK细胞的增殖率;流式细胞术(FCM)检测各组CIK细胞CD3+CD8+、CD3+CD56+、Treg表型比例;乳酸脱氢酶(LDH)释放法检测各组CIK细胞对肾癌786-O细胞的杀伤功能。结果磁珠分选后,PBMC中Treg细胞的比例降低至(1.21±0.53)%;PHA-IL-6-CIK组细胞的增殖率达(28.33±3.05)%;PHA-IL-6-CIK组细胞的CD3+CD8+、CD3+CD56+比例分别升高至(63.13±2.70)%和(35.32±2.96)%,Treg细胞比例降低至(1.11±0.07)%;在效靶比为30:1时,PHA-IL-6-CIK组细胞对肾癌786-O细胞的杀伤率可达(42.76±3.40)%。结论 IL-6、PHA联合磁珠分选可显著增强CIK的增殖和杀伤肾癌786-O细胞能力。

Objective To observe the effects of IL-6,PHA and IMB sorting on the proliferation of cytokine induced killer（ CIK） cells and the killing activity of CIK cells on renal cancer cells 786-O in vitro,and provide important theoretical basis for renal cancer immunotherapy. Methods Peripheral blood mononuclear cells（ PBMC） were isolated from the patients,CD4＋CD25＋Treg cells were removed with Mini MACS immunomagnetic bead（ IMB） sorting. PBMC were divided into control group（ cells were cultured in presence of CD3 Mc Ab,IL-2,IFN-γ）,PHA-CIK group（ cells were cultured in presence of CD3 Mc Ab,IL-2,IFN-γ and PHA）,IL-6-CIK group（ cells were cultured in presence of CD3 Mc Ab,IL-2,IFN-γand IL-6） and PHA-IL-6-CIK group（ cells were cultured in presence of CD3 Mc Ab,IL-2,IFN-γ,PHA and IL-6）. The proliferation rate of CIK cells of each group was assayed by Cell Counting Kit-8; flow cytometry（ FCM） was used to detect the proportion of phenotypes（ including CD3＋CD8＋,CD3＋CD56＋,Treg cells）. Lactate dehydrogenase（ LDH） release assay was used to measure cytotoxicity of CIK cells of each group against renal cancer cell 786-O. Results After IMB sorting,the proportion of Treg cells in PBMC decreased to（ 1. 21 ± 0. 53） %; the proliferation rate of PHA-IL-6-CIK cells was up to（ 28. 33 ± 3. 05） %; the proportion of CD3＋CD8＋and CD3＋CD56＋increased to（ 63. 13 ± 2. 70） % and（ 35. 32 ±2. 96） %; the proportion of Treg cells decreased to（ 1. 11 ± 0. 07） %; target ratio of 30∶ 1,and the killing rate of PHA-IL-6-CIK cells to renal cancer cells 786-O was up to（ 42. 76 ± 3. 40） %. Conclusion IL-6,PHA and IMB sorting could increase the proliferation of CIK cells and the killing activity of CIK cells on renal cancer cells in vitro significantly.