大鼠5-羟色胺1A受体超表达细胞株的构建

Construction of cell line with overexpression of rat serotonin1A receptor

目的 构建大鼠5-羟色胺1A受体（5-HT1AR）超表达细胞株.方法 从雄性SD大鼠脑组织中提取总RNA,构建真核表达质粒pc-DNA3.1/hisC-Rat-5-HT1AR.提取和纯化该真核表达质粒,并采用脂质转染法转染到人神经母细胞瘤细胞SH-SY5Y细胞.经G418筛选,获得SH-SY5Y-Rat-5-HT1AR细胞株.采用Western blot法鉴定5-HT1AR蛋白表达;光镜下观察细胞的形态;四甲基偶氮唑盐比色法测定细胞活力;细胞免疫荧光染色后,共聚焦显微镜下观察5-HT1AR表达.结果 成功构建了重组质粒pc-DNA3.1/hisC-Rat-5-HT1AR.重组质粒顺利转染SH-SY5Y细胞,构建了稳定表达大鼠5-HT1AR的细胞株SH-SY5Y-Rat-5-HT1AR.SH-SY5Y-Rat-5-HT1AR细胞呈梭状,细长,突起长而少.SH-SY5Y-Rat-5-HT1AR细胞的活力明显低于SH-SY5Y细胞.共聚焦显微镜下5-HT1AR主要在细胞膜表达.结论 成功地构建了大鼠5-HT1AR超表达细胞株.

Objective To establish a cell line with overexpression of rat serotonin1A receptor （5-HT1AR）.Methods Human neuroblastoma cells-SH-SY5Y were donated by cancer institute attached to the 4 th Affiliated Hospital, Hebei Medical University. Total RNA was extracted from brain tissues of male SD rats and rat 5-HT1A R was obtained by RT-PCR. Plasmid pc-DNA3. 1/hisC containing the rat 5-HT1AR （pc-DNA3.1/hisC-Rat-5-HT1AR）was constructed and transfected into SH-SY5Y cells. The transfected cells were isolated by G418 selection and SH-SY5Y-Rat-5-HT1A R cells were obtained. Expression of 5-HT1A R was detected by Western blot analysis. Cell viability was evaluated by MTT assay. SH-SY5Y-Rat-5-HT1AR cells were further observed for 5-HT1AR by immuno-fluorescence staining. Results Plasmid pc-DNA3. 1/hisC-Rat-5-HT1AR was successfully constructed by linking Rat-5-HT1A R with pc-DNA3.1/hisC and transfected into SH-SY5Y. The SH-SY5Y-Rat-5-HT1A R cells were more slender than SH-SY5Y cells with less and longer processes. MTT showed that the viability of SH-SY5Y-Rat-5-HT1A R cells was much lower than SH-SY5Y. Rat 5-HT1A R was expressed efficiently on the membrane of SH-SY5Y-Rat-5-HT1A R cells. Conclusion A cell line with overexpress of rat 5-HT1A R is successfully established.