稳定高表达ANO1对Hep-2细胞生物学性状的影响

Effects of stable ANO1 overexpression on biological characters of Hep-2 cells

目的:建立外源性ANO1稳定高表达的Hep-2细胞株,并观察其对Hep-2细胞生物学性状的影响。方法:设计引物,提取头颈鳞癌患者的临床标本DNA,并以其为模板行PCR,得到ANO1基因序列片段,将ANO1片段插入到PSG-5质粒,转染入Hep-2细胞株,将稳定高表达ANO1的Hep-2细胞株设为实验组,空白质粒转染的Hep-2细胞株设为对照组。并利用Western blotting法和免疫荧光实验检测已转染细胞,观察ANO1的表达情况。利用Boyden小室侵袭实验和黏附实验检测Hep-2细胞生物学性状的变化。结果:成功构建含ANO1片段的pSG-5质粒,Western blotting法检测结果显示,各组均出现目的条带,实验组的相对分子质量为100 000处条带明显较浓。间接免疫荧光实验结果显示,稳定高表达ANO1的Hep-2细胞内显现出亮绿色荧光,而空白质粒转染的Hep-2细胞内仅有较暗、较浅的绿色荧光。Boyden小室侵袭实验和黏附实验结果显示,稳定高表达ANO1的Hep-2细胞穿膜细胞百分比和黏附细胞百分比均明显高于对照组,两组结果比较差异有统计学意义(P<0.05)。结论:成功建立了ANO1稳定高表达的Hep-2细胞株,稳定高表达ANO1提高了Hep-2细胞的迁移能力。

Objective To establish human squamous carcinoma cell line Hep-2 stably overexpressing exogenous ANO1,and to observe its effects on the biological characters of Hep-2 cells.Methods Primers were designed.DNA was extracted from clinical specimens of patients with head and neck squamous cell carcinoma,and taken as template for PCR.Gene sequences of ANO1 fragment were gotten.ANO1 fragment was inserted into the pSG-5 plasmid,which was transfected into Hep-2 cells.These Hep-2 cell lines with ANO1 stable overexpression were regarded as experiment group,and those Hep-2 cell lines transfected with blank plasmid were regarded as control group.The expression of ANO1 was detected by Western blotting and immunofluorescence assay in the Hep-2 cells after transfection.Boyden chamber invasion assay and adhesion assay were used to observe the effects of ANO1 overexpression on the biological characters of Hep-2 cells.Results The PSG-5 plasmid containing ANO1 fragments was successfully constructed.The expression of ANO1 in Hep-2 cell line after transfection was observed by Western blotting.The result of Western blotting showed that the target band occurred in each group,and 100 000 band was darker in experiment group.Indirect immunofluorescence assay showed that the bright green fluorescence presented in Hep-2 cells with stable overexpression of ANO1;while darker,lighter green fluorescence in Hep-2 cells after transfection with the blank plasmid.The percentage of ANO1 overexpressing Hep-2 cells migrating through the membrane and percentage of ANO1 overexpressing Hep-2 cells adhesion of cells were greater than those in control group（P0.05）.Conclusion The Hep-2 cell line with ANO1 stable overexpression is successfully established.Stable ANO1 overexpression can improve the migration of Hep-2 cells.