自剪切多肽P2A对自然杀伤细胞的高效诱导扩增作用的研究

doi:10.3877/cma.j.issn.2095-1221.2019.05.005

目的

改进现有过继性人自然杀伤（NK）细胞体外诱导、分化及扩增方法，实现高效定向分化及大量扩增NK细胞的目的。

方法

利用自剪切多肽P2A连接IL-15和4-1BBL基因，通过慢病毒感染和药物筛选的方法制备稳定表达IL-15和4-1BBL的K562滋养层细胞株。该方法可刺激人PBMC细胞向NK细胞分化及高效增殖。实验结果以±s表示并用两样本t检验进行比较。

结果

经该方法培养的CD3^- CD16⁺ 56⁺ NK细胞增殖倍数达到（4480.43±37.80）倍；CD3^- CD16⁺ 56⁺ NK细胞纯度达到94.79%；细胞内表达IFN-γ和TNF-α的NK细胞比例为（63.07±3.37）%和（54.85±2.04）%，分别高于对照组（16.28±2.86）%和（14.53±1.15）%（t = 62.25，22.66，P均< 0.01）；细胞分泌至培养上清液中的IFN-γ和TNF-α浓度为（111.39±6.95）?pg/ml和（32.76±3.23） pg/ml，分别高于对照组（44.99±4.74）pg/ml和（11.09±2.45）?pg/ml（t = 20.56，7.21，P均< 0.01）；在体外，该方法培养的NK细胞对K562细胞的杀伤效率为（78.52±7.36）%，高于对照组（48.53±6.66）%（t = 11.56，P < 0.01）；对H520细胞的杀伤效率为（65.03±3.27）%，高于对照组（35.85±3.99）%（t = 11.35，P < 0.01）。

结论

利用自剪切多肽P2A构建稳定高表达的IL-15和4-1BBL的K562滋养层细胞株，从而提高了NK细胞增殖倍数、纯度和细胞毒性。

关键词: 自剪切多肽P2A, 自然杀伤细胞, 滋养层细胞, 细胞免疫治疗

Objective

The purpose of this study was to improve differentiation and expansion for nature killer cells in vitro using self-cleaving P2A peptide.

Methods

IL-15 and 4-1BBL genes were connected via P2A. Lentiviral vector and drug screening were used to produce K562 feeder cells which were stably expressing IL-15 and 4-1BBL. Subsequently, they were co-cultured with human PBMC as feeder cells for NK cell differentiation and proliferation. The results were expressed as ±s and compared by two-sample t-test.

Results

The fold of CD3^-CD16⁺56⁺ NK cell expansion was 4480.43+37.80 times in vitro. The proportion of CD3^-CD16⁺56⁺ NK cells was 94.79%. The percentage of IFN-γ and TNF-α positive NK cells was (63.07±3.37) % and (54.85±2.04) %, respectively, which were higher than that of the control group (16.28±2.86) % and (14.53±1.15) %, (t = 62.25 and t = 22.66 respectively, P < 0.01) . The concentrations of IFN-γ and TNF-α secreted by NK cells were (111.39±6.95) pg/ml and (32.76±3.23) pg/ml, respectively, and higher than that of the control group (44.99±4.74) g/ml and (11.09±2.45) pg/ml, (t = 20.56 and t = 7.21 respectively, P < 0.01) . In vitro, the killing rates of NK cells against K562 and H520 cells were (78.52±7.36) % and (65.03±3.27) %, respectively, which were higher than those of the control group (48.53±6.66) % and (35.85±3.99) %, t = 11.56 and t = 11.35 respectively, P < 0.01) .

Conclusion

We have successfully improved the transmembrane expression of IL-15 and 4-1BBL in K562 feeder cells, and established a better culture method to obtain NK cells from human PBMCs.

Key words: Self-cleaving P2A peptide, Natural killer cells, Feeder cells, Immunotherapy

图1 pLVX-IL15-41BBL慢病毒表达载体的构建

图2 pLVX-IL15-41BBL质粒转化克隆PCR产物的电泳条带

图3 梯度稀释的ZsGreen1慢病毒感染293T细胞的流式检测

图4 K562细胞内目的蛋白自剪切及跨膜表达

图5 慢病毒感染前后IL15-41BBL+ K562细胞的流式检测

图6 倒置显微镜下观察经滋养层细胞诱导培养16 d后获得的两组自然杀伤细胞（明场，×100）

图7 两组自然杀伤细胞诱导培养16 d的生长曲线

图8 外周血单个核细胞及诱导培养16 d的两组自然杀伤细胞的细胞表型流式检测

图9 CD3^- CD56⁺自然杀伤细胞的增殖倍数比较

图10 两组自然杀伤细胞内γ-干扰素、α-干扰素细胞因子表达的流式散点检测

图11 两组自然杀伤细胞内γ-干扰素、α-干扰素细胞因子表达阳性比例

图12 两组自然杀伤细胞培养上清液中γ-干扰素、α-干扰素细胞因子的流式散点检测

图13 两组自然杀伤细胞培养上清液中γ-干扰素、α-干扰素细胞因子浓度的比较

图14 两组自然杀伤细胞对肿瘤细胞系K562和H520细胞的杀伤比例

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Application of self-cleaving P2A peptide for the expansion of natural killer cells

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Application of self-cleaving P2A peptide for the expansion of natural killer cells