负载肺癌干细胞膜微粒的DC-CIK对耐药肺癌细胞的靶向杀伤作用及对肺癌干细胞凋亡的影响

Killing effect of DC-CIK cells loaded with MMPs on EGFR-TKI-resistant lung cancer cells and the apopotosis inducing effect on lung cancer stem cell

目的:探讨负载肺癌干细胞膜微粒的DC-CIK细胞对EGFR-TKI耐药肺癌细胞的杀伤作用及对肺癌干细胞凋亡的影响机制。方法:无血清悬浮细胞培养法富集EGFR-TKI耐药肺癌细胞A549、H292干细胞样细胞,RT-PCR检测干细胞标志物,裸鼠成瘤实验鉴定致瘤性。超滤和差速离心法获取肺癌干细胞膜微粒。流式细胞术分别测定共孵育组和常规培养组DC成熟标志CD86和CD83,测定两组DC-CIK细胞表型CD3^+、CD3^+CD8^+、CD3^+CD56^+、CD3^+CD4^+;形态观察及MTT法分别测定不同效靶比两组DC-CIK对A549、H292的杀伤效应;ELISA法分别检测两组DC-CIK上清中IL-2、IFN-γ、TNF-α分泌水平;流式细胞术分别测定两组DC-CIK对肺癌干细胞凋亡的影响。结果:富集培养获得的EGFR-TKI耐药肺癌干细胞样细胞高表达干细胞标志物Sox2和Oct4,并具较强裸鼠致瘤性。负载膜微粒的DC成熟标志CD86和CD83较常规DC表达显著升高;负载膜微粒的DC-CIK较常规DC-CIK细胞表型CD3^+、CD3^+CD8^+、CD3^+CD56^+、CD3^+CD4^+升高,对EGFR-TKI耐药肺癌细胞杀伤效应高于常规DC-CIK,并具有显著提高的靶向趋向性;负载膜微粒的DC-CIK分泌因子对EGFR-TKI耐药肺癌干细胞样细胞凋亡的影响与常规DC-CIK不同。结论:与常规培养DC-CIK相比,负载膜微粒的DC-CIK活性提高,对EGFR-TKI耐药肺癌细胞的体外特异靶向杀伤效应显著提高。细胞分泌因子可显著上调耐药肺癌干细胞的细胞凋亡率。

Objective： To investigate the target killing effect of dendritic cells（ DC）- cytokine- induced killer（ CIK） cells loaded with membrane- based microparticles（ MMPs） derived from lung cancer stem cells on EGFR-TKI- resistant lung cancer cells,and apopotosis inducing effect on lung cancer stem cell and to explore its potential mechanisms. Methods： Human lung cancer EGFR tyrosine kinase inhibitor（ EGFR- TKI） resistant A549,H292 stem cells were isolated and cultured in serum- free medium,the expression of Sox2 and Oct4 was detected by reverse transcription- polymerase chain reaction（ RT- PCR）. The tumor- inducing ability of lung cancer stem cells was identified by xenografts in nudemice. MMPs were isolated by differential centrifugation and ultrafiltration from lung cancer A549,H292 stem cells. DC and CIK cells were induced from the peripheral blood mononuclear cells of healthy volunteers and some of the DC cells loaded with MMPs were used as loaded group,the other DC cells stimulated with TNF- α were used as control group. The phenotype CD83,CD86 of DC cells in each group was examined respectively with flow cytometry（ FCM）. After the DC cells in each group were co- cultured respectively with CIK cells for 6 days,the phenotype of CD3^＋,CD3^＋CD8^＋,CD3^＋CD56^＋,CD3^＋CD4^＋ of DC- CIK cells in each group was detected respectively with FCM. The killing effect on EGFR- TKI- resistant lung cancer cells of DC- CIK cells in each group was detected by methyl thiazolyl tetrazolium（ MTT）. The level of IL- 2,IFN- γ,TNF- α in suspention of DC- CIK cells was detected by ELISA. The apopotosis of EGFR- TKI- resistant lung cancer stem cell induced by the suspention of DC- CIK cells in each group was examined by FCM. Results： The surface maturity markers CD83,CD86 of DC cells in loaded group significantly increased. Meanwhile,the percentages of CD3^＋CD8^＋ cells and CD3^＋CD56^＋ cells of DC- CIK cells in loaded group went up significantly compared with the controls. The tumor tropism of DC-CIK cells in loaded group significantly increased. MTT test and the observation by inverted microscope showed that the target destruction of DC- CIK cells in loaded group significantly increased. The level of IL- 2,IFN- γ,TNF- α in suspention of DC- CIK cells in loaded group was higher than in control group. The apopotosis of EGFR- TKI- resistant lung cancer stem cell induced by the suspention of DC- CIK cells in loaded group significantly increased.Conclusion： The immunofunction of DC cells could be enhanced by loading with MMPs derived from A549,H292 stem cells. The coculture of CIK and DC cells loaded with MMPs could promote CIK cell immunoreactions,proliferation,and improve target killing activity to EGFR- TKI- resistant lung cancer cells and induce apopotosis of lung cancer stem cells.