不可逆电穿孔对结肠癌细胞疗效和作用机制的体内外实验研究

Effect and mechanism of irreversible electroporation on colon cancer cells in vivo and in vitro

目的探讨不可逆电穿孔(IRE)在体内外模型中对结肠癌细胞生长抑制的作用机制及有效性。方法对不同分化程度的结肠癌细胞株HCT116、SW480、SW620、LoVo进行电击处理,通过二乙酸荧光素(FDA)-碘化丙锭(PI)双色荧光染色实验检测细胞活性,膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/PI双染流式细胞术检测细胞凋亡,蛋白质印迹法(Western blot)检测凋亡标志蛋白表达;构建HCT116细胞裸鼠异种移植瘤模型,通过裸鼠体重、肿瘤生长曲线、瘤重对比观察体内抗结肠癌效果。采用非配对t检验。结果FDA-PI荧光实验显示,随着场强增大,SW480、HCT116均表现为细胞总量、活细胞数减少,死细胞数增多。流式双染实验显示,随着场强增大,凋亡细胞比例逐渐增多,多数为早期凋亡。Western blot结果为电击后多聚二磷酸腺苷核糖聚合酶降解产物(cleaved-PARP)和半胱氨酸蛋白酶3,7,8,9裂解物(cleaved-Caspase 3,7,8,9)蛋白水平升高。IRE组与Control组裸鼠体重分别为(27.81±0.81)、(27.64±0.83)g(t=0.583,P>0.05);IRE组与Control组肿瘤体积分别为(212.00±98.85)、(4381.75±910.20)mm^(3)(t=7.713,P<0.001);IRE组与Control组第15天瘤重分别为(79.93±15.41)、(1500.97±848.51)mg(t=2.900,P<0.05)。结论IRE能有效抑制结肠癌细胞生长。

Objective To evaluate the mechanisms and effectiveness of irreversible electroporation(IRE)on the growith inhibition of colon cancer cells in both in vivo and in vitro models.Methods We first treated colon cancer cell lines with different levels of differentiation such as HCT116,SW480,SW620 and LoVo with IRE,then detected cell activity by fluorescein diacetate(FDA)-propidium iodide(PI)two-color fluorescence staining,cell apoptosis by annexin V-fluorescein isothiocyanate(Annexin V-FITC)/PI double staining flow cytometry,and expression levels of apoptotic marker proteins by Western blotting.Nude mice model subcutaneously inoculated with HCT116 cells were established to explore the inhibitory effect of IRE on colon tumor growth by comparing body weights,tumor growth curves and tumor weights.Results As revealed by the FDA-PI fluorescence staining experiments,IRE led to the decreased number of total and living cells,and increased number of dead cells.Flow cytometry showed that the proportion of apoptotic cells increased with the rising electric field intensity,and most of them are early apoptotic cells.Moreover,we uncovered that IRE induced SW480 cell apoptosis through mitochondria and death receptor-dependent pathways,as evidenced by increased expression of cleaved-poly ADP-ribose polymerase(cleaved PARP)and cleaved Caspase 3,7,8,9.The body weights in nude mice of the IRE group and the control group were(27.81±0.81)g and(27.64±0.83)g(t=0.583,P>0.05),respectively.The tumor volumes in the IRE group and the control group were(212.00±98.85)mm^(3)and(4381.75±910.20)mm^(3)respectively(t=7.713,P<0.01).The tumor weights in the IRE group and the control group on the day 15 were(79.93±15.41)mg and(1500.97±848.51)mg(t=2.900,P<0.05),respectively.Conclusion IRE can effectively inhibit the growth of colon cancer cells and is expected to be applied in the treatment of colon cancer combined with endoscopic technology in the future.