LSD1过表达及去甲基化作用缺失质粒的构建与鉴定

Construction and identification of LSD1 overexpression and demethylation disfunction plasmids

目的 评估赖氨酸特异性组蛋白去甲基化酶1（LSD1）过表达质粒和LSD1去甲基化片段缺失质粒在人胚肾细胞HEK293T中的表达水平。方法 采用PCR扩增LSD1片段，重叠PCR扩增LSD1去甲基化作用缺失片段，构建大鼠特异性LSD1过表达质粒和LSD1去甲基化片段缺失质粒，并经琼脂糖凝胶电泳和测序进行验证。将验证后的两种重组质粒与空白载体分别进行包病毒，感染HEK293T后用嘌呤霉素筛选稳定表达的细胞，再用Western Blot和实时定量PCR检测稳定表达的HEK293T细胞内LSD1的表达情况。结果 琼脂糖凝胶电泳和测序结果表明，LSD1过表达质粒和LSD1去甲基化片段缺失质粒构建成功。Western Blot和实时定量PCR检测结果显示，与空白对照组相比，LSD1过表达组和LSD1去甲基化片段缺失组HEK293T细胞中LSD1蛋白及mRNA的相对表达量均上调，差异均具有统计学意义（均P〈0.01）。结论 构建的LSD1过表达质粒和LSD1去甲基化片段缺失质粒在HEK293T细胞中实现了LSD1基因的过表达，为进一步研究LSD1基因与相关疾病的关系以及LSD1去甲基化作用奠定了基础。

Objective To construct rat lysine-specific histone demethylase 1（LSD1）overexpression plasmids and LSD1 demethylation fragment disfunction plasmids,and to evaluate their expression levels in HEK293T cells.Methods LSD1 fragments were amplified by PCR,and LSD1 demethylation disfunction fragments were amplified by overlap PCR.Rat-specific LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were constructed and verified by agarose gel electrophoresis and sequencing.HEK293T cells were infected using the validated recombinant plasmids and blank vectors,and the stably expressed cells were selected by puromycin.The expression of LSD1 in the stably expressed HEK293T was detected by Western Blot and real-time quantitative PCR.Results The results of agarose gel electrophoresis and sequencing showed that LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were successfully constructed.The Western Blot and real-time quantitative PCR results showed that compared with the blank group,the relative expression of LSD1 and mRNA in the LSD1 overexpression group and the LSD1 demethylation disfunction group were up-regulated,and the differences were statistically significant（all P〈0.01）.Conclusions The constructed LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids can achieve overexpression of LSD1 gene in HEK293T cells.This paper lays a foundation for further study of the relationship between LSD1 gene and related diseases and demethylation of LSD1.