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

摘要:

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pEGFP-N1-LINE1-ORF1，转染WT_CLS1细胞，通过Western blot和qRT-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pEGFP-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。

Abstract:

Objective To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells. Methods A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p 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-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone. Results The LINE1-ORF1p antibody prepared had a titer of > 1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA 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.