Dishevelled促进脑胶质瘤增殖的实验研究

Dishevelled promotes proliferation of glioma:An experimental study

目的探讨Dishevelled表达对脑胶质瘤增殖能力的实验研究。方法采用蛋白质免疫印迹法(Western blot)和实时定量反转录聚合酶链反应(RT-PCR)筛选出3株脑胶质瘤细胞系(U373、U251和U87)中Dishevelled表达水平最高的脑胶质瘤细胞系,通过慢病毒转染技术使Dishevelled低表达,构建出低表达Dishev-elled的脑胶质瘤细胞模型。采用细胞增殖生物因子活性检测(CCK)-8实验,观察Dishevelled低表达对脑胶质瘤细胞体外增殖能力的影响。构建荷瘤鼠模型,采用小动物活体成像观察Dishevelled低表达对脑胶质瘤体内增殖能力的影响。结果RT-PCR和Western blot检测发现,Dishevelled蛋白在3株脑胶质瘤细胞系中均过表达,且在脑胶质瘤U251细胞中表达水平最高;采用慢病毒转染技术敲低Dishevelled的表达,应用CCK-8实验结果显示:a组(Dishevelled敲低)、b组(无关序列)、c组(正常U251细胞)的A 450值分别为:1.41±0.08;2.51±0.04;2.74±0.07。其中a组明显低于b、c组,差异具有统计学意义(P=0.032),而b、c组无明显差异(P=0.834)。应用小动物活体成像结果显示:a组(Dishevelled敲低)、b组(无关序列)、c组(正常U251细胞)颅内肿瘤荧光强度分别为:175415±60178;367328±56014;449892±66980。a组荧光强度明显低于b组(P=0.041)和c组(P=0.045),而b、c组之间无明显差异(P=0.745)。这表明,低表达Dishevelled可显著下调脑胶质瘤体外和体内增殖能力。结论Dishevelled在脑胶质瘤中高表达,并可促进脑胶质瘤增殖,与脑胶质瘤的发生发展密切相关。

Objective To investigate the effect of Dishevelled expression on the proliferation of glioma.Methods Three glioma cell lines(U373,U251,and U87)were screened by Western blot and RT-PCR,from which the glioma cell line with highest Dishevelled expression was selected.Low expression of Dishevelled was achieved through lentiviral transfection,and thereby a glioma cell model with low Dishevelled expression was constructed.Cell proliferation(CCK-8)assay was used to examine the effect of low Dishevelled expression on proliferation of glioma cells in vitro.A tumor-bearing mouse model was established.In-vivo small-animal imaging was used to determine the effect of low Dishevelled expression on proliferation of glioma in vivo.Results RT-PCR and Western blot detection showed that Dishevelled protein was overexpressed in all of the three glioma cell lines,and the expression level was highest in glioma U251 cells.Lentiviral transfection was used to knock down the Dishevelled expression.After this,CCK-8 assay showed that the A450 values of group A(Dishevelled knockdown),group B(irrelevant sequence),and group C(normal U251 cells)were 1.41±0.08,2.51±0.04,and 2.74±0.07,respectively.Among them,the A 450 value was significantly lower in group A compared with groups B and C,with statistically significant difference(P=0.032);but there was no significant difference between the latter two groups(P=0.834).In vivo small animal imaging showed that the fluorescence intensity of intracranial tumors in group A(Dishevelled knockdown),group B(irrelevant sequence),and group C(normal U251 cells)were 175415±60178;367328±56014;and 449892±66980,respectively.The fluorescence intensity was significantly lower in group A compared with group B(P=0.041)or group C(P=0.045),but there was no significant difference between the latter two groups(P=0.745).These findings suggested low expression of Dishevelled might significantly down-regulate the proliferation of glioma in vitro and in vivo.Conclusion Dishevelled is highly expressed in gliomas.It may promote the proliferation of gliomas,and is thus closely related to development and progression of gliomas.