CnAβ基因对大鼠RBL-2H3细胞脱颗粒的影响

Effect of CnAβGene on Degranulation of Rat RBL-2H3 Cells

过敏是人和养殖动物常见的疾病,而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制,本研究利用CRISPR/Cas9基因编辑技术,构建钙调磷酸酶A beta(calcineurin A beta,CnAβ)基因敲除的大鼠RBL-2H3细胞株,并探讨CnAβ基因对RBL-2H3细胞生长及脱颗粒的影响。选取大鼠CnAβ基因第一外显子为敲除靶点,设计并合成3个单导向RNA(single guide RNA,sgRNA),构建pX459-CnAβ-sgRNA质粒,并用脂质体3000将构建好的质粒转染到RBL-2H3细胞内;利用嘌呤霉素对转染细胞进行筛选,通过DNA测序验证获得CnAβ基因敲除的RBL-2H3细胞株;并检测CnAβ基因缺失对细胞增殖和脱颗粒的影响。结果表明,成功构建了CnAβ单基因敲除的大鼠RBL-2H3细胞株;CnAβ基因缺失对RBL-2H3细胞增殖、细胞的颗粒形成以及颗粒含量无显著影响,但显著抑制由细胞表面受体介导的RBL-2H3细胞脱颗粒作用。该研究结果有助于深入了解动物过敏性疾病的发生机制,为动物过敏性疾病的预防提供了理论基础。

Allergy is a common disease in humans and farmed animals,and it develops quickly.To insight into the molecular mechanism of allergic disease,this study was focused on the function of calcineurin A beta(CnAβ)gene on RBL-2H3 cell degranulation.CRISPR/Cas9 gene editing technology was employed to construct CnAβgene knockout RBL-2H3 cell line,and the proliferation and degranulation of RBL-2H3 cell effected by CnAβgene were surveyed.The first exon of rat CnAβgene was selected as a knockout target for the design and synthesis of three single guide RNAs(sgRNA),to construct the pX459-CnAβ-sgRNA knockout plasmid,and the constructed plasmid was transferred into RBL-2H3 cells by Lipofectamine 3000.After puromycin screening,the RBL-2H3 cell line with CnAβgene deletion was obtained by PCR and DNA sequencing,and then used to detect the effect of CnAβgene deletion on cell proliferation and degranulation.The results indicated that CnAβgene knockout RBL-2H3 cell line was successfully constructed,and CnAβdeficiency had no significant effect on the proliferation,granule formation or granule content of RBL-2H3 cell,but significantly inhibited the degranulation of RBL-2H3 cells mediated by cell surface receptors.This research will contribute to an in-depth understanding of the mechanism of animal allergic diseases and provide a theoretical basis for the prevention of allergic disease.