LINE1-ORF1p过表达对肾母细胞瘤细胞WT_CLS1增殖的影响

Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells

目的制备LINE1-ORF1p多克隆抗体,研究LINE1-ORF1p过表达对肾母细胞瘤细胞WT_CLS1增殖的影响。方法利用基因工程方法原核表达LINE1-ORF1p,免疫家兔制备多克隆抗体。间接ELISA法检测抗体效价,通过Western?blot及免疫组化方法检测抗体对LINE1-ORF1p的特异性识别能力。构建真核表达载体p EGFP-N1-LINE1-ORF1,转染WT_CLS1细胞,通过Western?blot和q RT-PCR检测LINE1-ORF1蛋白和基因的表达情况,采用细胞增殖实验和平板克隆形成实验检测LINE1-ORF1p对WT_CLS1细胞增殖及肿瘤细胞克隆形成的影响。结果制备的LINE1-ORF1p抗体效价>1?:?16?000,能对细胞及肿瘤组织内LINE1-ORF1p特异识别。转染p EGFP-N1-LINE1-ORF1的WT_CLS1细胞,其LINE1-ORF1的mRNA及蛋白水平显著增高(P<0.05),细胞增殖能力和克隆形成能力都显著增强(P<0.05)。结论 LINE1-ORF1p可以促进肾母细胞瘤细胞的生长和肿瘤细胞克隆形成,可能参与肾母细胞瘤的发病机制。

Objective To prepare the LINE1-ORF1 p polyclonal antibody, and to study the effect of LINE1-ORF1 p on the proliferation of nephroblastoma WT_CLS1 cells. Methods A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1 p, and rabbits were immunized with LINE1-ORF1 p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1 p. The eukaryotic expression vector p EGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and q RT-PCR were used to measure the protein and m RNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1 p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone. Results The LINE1-ORF1 p antibody prepared had a titer of 〉1：16 000 and could specifically recognize LINE1-ORF1 p in cells and tumor tissue. WT_CLS1 cells transfected with p EGFP-N1-LINE1-ORF1 had significant increases in the m RNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability（P〈0.05）. Conclusions LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.