长期培养的CIK细胞hTERT基因表达水平的动态变化

摘要

Objective; To investigate the dyhamic changes of the expression of human telomerase reverse tran-scriptase ( hTERT) in the long - term cultured CIK cells, and evaluate the safety of CIK cells. Methods; Peripheral blood mononuclear cells(PBMCs) from 3 healthy donors were incubated to induce CIK cells. Cell proliferation was measured weekly by counting viable cells based on trypan blue exclusion. The hTERT mRNA expression levels of CIK cells were analyzed by quantitative PCR on day 0, 2, 7, 21, and 42 during the incubation period. Cell cycle and ap-optosis were analyzed by flow cytometry on day 0,7, 14,28,49 and 56 during the incubation period, cytotoxicity of CIK cells against LTE cell line was determined by MTT assays. Results: The maximum proliferation of CIK reached at the 28nd and the 35nd day,the total CIK cells significantly increased by 6. 7 to 15. 95 folds in cell proliferation number compared with cells cultured on day 0. Their viability decreased progressively until 2 months, when almost all CIK cells were dead. The hTERT expression was very low in quiescent lymphocytes, rapidly increased to reach a maximum after 2 days of culture, and roughly decreased to its basal level, and remained at a low level. At 7 days, the percentages of CIK cells in S phase was (41.27 ±4.57)% , at 49 days most of the cells were in G1/G0 phase. The mean percent specific lysis of CIK cells decreased from (58.58 ±8.52)% at the day 14 to (32.47 ±7.51)% at the day 30. Conclusion: The hTERT expression decreased to a low level during the in vitro culture of the CIK cells, the CIK cells were not immortalized during the long - term culture and repeated stimulations.%目的:初步探讨长期培养的CIK细胞hTERT基因表达水平的变化,进一步评价CIK细胞治疗的安全性.方法:抽取3名健康人外周静脉血进行CIK细胞培养,每周台盼蓝染色计数细胞,实时荧光定量PCR法检测培养第0、2、7、21及42天的CIK细胞hTERT基因的表达水平,流式细胞仪分析培养第0、7、14、28、49及56天的CIK细胞周期的改变并检测凋亡,MTT法测定培养第14、30天的CIK细胞对肺癌LTE细胞系的细胞毒活性.结果:CIK细胞在体外培养第28天至第35天细胞增殖达高峰,与第0天相比扩增约6.7至15.95倍,此后存活细胞逐渐减少,最终约60天左右细胞全部死亡,与流式细胞仪检测细胞凋亡结果一致.hTERT表达水平在培养48小时最高,较第0天增加约(2.84±1.27)倍,随后随培养时间延长表达水平下降,在培养1周时降至培养初始水平,并在此后的培养过程中维持低水平表达.在CIK培养1周时处于S期细胞比例最高,约(41.27±4.57)％,随培养时间延长Go/G1期细胞比例占绝大部分,在培养第49天Go/G1期细胞比例在90％以上[(97.56±1.17)％]；CIK细胞在第14天杀瘤活性约(58.58±8.52)％,在30天时杀瘤活性明显下降,约(32.47±7.51)％.结论:长期培养的CIK细胞随培养时间延长hTERT表达水平下降,未见细胞永生化倾向.