肝癌干细胞的培养、鉴定及CIK对其生长、增殖的影响

Effect of cytokine induced killer on the growth and proliferation of liver cancer stem cells

摘要:

目的：探讨细胞因子诱导的杀伤细胞（cytokine induced killer，CIK）对肝癌干细胞（liver cancer stem cells，LCSCs）生长、增殖的影响。方法：用含多种细胞因子（LIF、EGF、bFGF及B27）的无血清培养液（serum- free medium，SFM）诱导SMMC7721人肝癌细胞产生LCSCs，并行LCSCs表面标志CD133、CD90的流式仪（flow cytometry，FCM）检测鉴定与裸鼠致瘤能力观测。以IFN-γ、CD3单克隆抗体（human CD3 monoclonal antibody，hCD3mAb）与 IL-2等诱导肝癌患者外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）的悬浮细胞生成CIK。用Transwell培养小室将LCSCs与不同密度的CIK在同一培养体系内以该小室膜分隔培养24、48、72 h，采用CCK-8试剂盒检测CIK对LCSCs生长、增殖的影响。以RT-PCR与Western blot分别检测与CIK共培养的LCSCs的增殖细胞核抗原（proliferating cell unclear antigen，PCNA）基因及其编码蛋白的表达变化。结果：FCM检测显示，LCSCs高表达干细胞表面标志分子CD133、CD90。CCK-8法检测表明，与对照比较，与相同密度的CIK共培养的LCSCs，其生长、增殖减慢，48 h开始变得更加明显（P<0.01）。RT-PCR、Western blot检测显示，CIK能明显下调LCSCs的PCNA基因及其编码蛋白的表达。结论：成功诱导培养并鉴定到LCSCs。肝癌患者的CIK可明显抑制LCSCs的生长、增殖与下调其PCNA基因及其编码蛋白的表达。

Abstract:

Objective：To explore the effect of cytokine induced killer（CIK） on the growth and proliferation of liver cancer stem cells （LCSCs）. Methods：LCSCs were prepared from liver cancer cell line SMMC7721 induced by serum-free medium（SFM） containing several cytokines（LIF，EGF，bFGF and B27），and their CD90 and CD133 were identified by flow cytometry（FCM）. Meanwhile，CIK cells were produced from suspended peripheral blood mononuclear cells（PBMCs） of patients with hepatocellular carcinoma（HCC） in－duced by IFN-γ，human CD3 monoclonal antibody（hCD3mAb） and IL-2. LCSCs and CIK were cultured in a same circumstance with a Transwell culturing cabin，but the LCSCs were cultured at one side of the membrane of the Transwell，and the CIK cells were cul－tured at another side of the same membrane for 24 h and 48 h respectively. The effects of CIK cells on the growth and proliferation of LCSCs were detected by cell count kit-8（CCK-8）. The expression of gene and encoding protein of proliferating cell nuclear antigen （PCNA） were assayed via RT-PCR and Western blot respectively. Results：The high expressions of CD90 and CD133 in LCSCs were tested by FCM. The results of CCK-8 indicated that the growth and proliferation of LCSCs co-cultured with the CIK cells were slower than those of control cells，which become more visible at 48 h after co-culturing（P<0.01）. The results of RT-PCR and Western blot revealed that CIK cells can significantly down-regulate the ex－pression of gene and encode protein of PCNA in LCSCs. Con－clusion：LCSCs are induced，cultured and identified successfully. The CIK cells induced from patients with HCC can significantly prevent LCSCs from growth and proliferation and down-regulate the gene and encode protein of PCNA in them.