摘要目的 探讨Latexin (Lxn)基因对CD133 MIAPaca-2胰腺癌干细胞增殖的影响.方法 采用磁珠分选法从MIAPaca-2胰腺癌细胞株中分选出CD133+细胞,在无血清培养基中观察其形态变化及种植到裸鼠皮下后的成瘤能力.采用脂质体法将不同浓度的插入Lxn基因(1、3、5μg)的质粒转染CD133+MIAPaca-2胰腺癌细胞.应用RT-PCR及蛋白质印迹法检测转染的癌细胞Lxn mRNA及蛋白表达量.CCK-8法检测转染癌细胞的增殖能力.结果 成功分离出CD13;MIAPaca-2胰腺癌细胞,它以悬浮成球的方式生长,以1 ×105个癌细胞种植裸鼠皮下的成瘤率达100％.转染插入Lxn的质粒后,CD133+MIAPaca-2胰腺癌细胞Lxn基因的表达呈转染剂量依赖性地增加,转染5μg质粒的细胞Lxn mRNA及蛋白表达量分别为20.80±0.98、16.80±2.73,显著高于未转染细胞的1.02 ±0.01、1.01 ±0.01,差异均有统计学意义(P值均＜0.05);转染后癌细胞增殖活性呈转染剂量及培养时间依赖性地被抑制,转染0.4 μg质粒的癌细胞的生长抑制率达36.2％,显著高于未转染细胞的抑制率,差异有统计学意义(P＜0.05).结论 CD133+ MIAPaca-2胰腺癌细胞具有肿瘤干细胞部分特性,Lxn基因转染对CD133+MIAPaca-2胰腺癌干细胞增殖具有抑制作用.

abstractsObjective To explore the effect of Latexin (Lxn) gene transfection on proliferation of CD13;MIAPaca-2 pancreatic cancer stem-like cells.Methods CD133+ MIAPaca-2 cells were isolated and sorted by magnetic activated cell sorting from pancreatic cancer MIAPaca-2 celt line.CD133+ MIAPaca-2 cells were cultured in serum-free medium and the capacity for proliferation,and tumorigenicity of CD133+ MIAPaca-2 cells was determined by the floating spheres test and tumor xenograft assays.The CD133+ MIAPaca-2 cells were transfected with Lxn plasmid (1,3,5 μg).After transfection,the protein and mRNA expression of Lxn in CD133+ and CD133+-MIAPaca-2 cells were detected by Western blotting and RT-PCR,respectively.Cell proliferation was assayed by CCK-8.Results CD133+ MIAPaca-2 cells were successfully isolated,and it grew into a ball-suspended way,the tumorigenicity rate in nude mice with subcutaneous injection 1 × 105 cancer cells was 100％.After Lxn plasmid transfection,the expression of Lxn in CD133+ MIAPaca-2 cells was increased in a dose dependent manner,the Lxn protein and mRNA expression of tumor cells transfected with 5 μg plasmid was 20.80 ±0.98,16.80± 2.73,which was significantly higher than that in non-transfected cells (1.02 ± 0.01,1.01 ± 0.01),and the difference between the two groups was statistically significant (P ＜ 0.05).After transfection,cellular proliferation activity also showed a transfection dose and culture time-dependent decrease,the inhibition rate of tumor cells transfected with 0.4 μg plasmid was 36.2％,which was significantly different from that in non-transfected cells (P ＜ 0.05).Conclusions CD133+ MIAPaca-2 pancreatic cancer cells have some characteristics of cancer stem cells.Lxn gene transfection can inhibit the proliferation of CD133+ MIAPaca-2 cells.