摘要：

目的: 探究基因间区长链非编码RNA-EPS（long intergenic noncoding RNA，lincRNA-EPS）对脂多糖（lipopolysaccharides，LPS）诱导的小鼠牙周膜细胞（mouse periodontal ligament cells，mPDLCs）炎症因子表达的影响。方法: 使用CRISPR/Cas9技术构建lincRNA-EPS基因敲除的C57BL/6J小鼠。分离培养野生型及lincRNA-EPS基因敲除型mPDLCs，并进行免疫荧光鉴定。制备纳米粒材料，搭载lincRNA-EPS过表达质粒，并测定转染效率。设立5个实验组：野生型对照组（WT组）、野生型刺激组（WT+LPS组）、敲除型对照组（KO组）、敲除型刺激组（KO+LPS组）和挽救实验组（KO+LPS+lincRNA-EPS组）。在WT+LPS组、KO+LPS组及KO+LPS+lincRNA-EPS组中添加100 ng/mL的LPS，刺激6 h，其中，在KO+LPS+lincRNA-EPS组中提前加入lincRNA-EPS过表达质粒进行转染。WT组及KO组不做处理。利用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction，RT-qPCR）检测各实验组炎症因子cxcl10、cxcl12、cxcl14、IL-1α、IL-6 mRNA的表达量。应用酶联免疫吸附测定技术（enzyme-linked immunosorbent assay，ELISA）测定各组炎症因子cxcl10、IL-1α、IL-6的蛋白水平。结果: 成功构建了lincRNA-EPS基因敲除的C57BL/6J小鼠模型；搭载lincRNA-EPS过表达质粒的纳米粒成功转染了mPDLCs，转染效率接近20%；在无LPS刺激的状态下，KO组的炎症因子表达水平较低，且与WT组无差异（P>0.05），lincRNA-EPS基因的缺失不会导致mPDLCs炎症因子基础表达量的改变；LPS刺激6 h后，KO+LPS组和WT+LPS组相较于各自的对照组，各个炎症因子的表达均上调（P<0.05），且KO+LPS组相较于WT+LPS组，各炎症因子表达上调更为显著（P<0.05），说明lincRNA-EPS缺失的mPDLCs受到LPS刺激时，更易引发强烈的炎症因子表达；KO+LPS+lincRNA-EPS组相较于KO+LPS组，炎症因子的表达量下调（P<0.05），可见外源性lincRNA-EPS的转入在一定程度上抑制了炎症因子的表达。结论: lincRNA-EPS对LPS诱导的mPDLCs炎症因子表达起负向调控作用。

关键词: 基因间区长链非编码RNA, 脂多糖, 牙周炎, 小鼠牙周膜细胞

Abstract:

Objective: To investigate the effects of long intergenic noncoding RNA-EPS (lincRNA-EPS) on the expression of inflammatory cytokines in lipopolysaccharides-induced (LPS-induced) mouse periodontal ligament cells(mPDLCs). Methods: To fabricate a C57BL/6J mouse model of lincRNA-EPS knockout with CRISPR/Cas9 technique. MPDLCs of wild-type mice and lincRNA-EPS knockout mice were isolated，cultured and verified by immunofluorescence technique. Nanoparticles were prepared and loaded with lincRNA-EPS overexpressed plasmid, and the transfection efficiency was determined. 5 experimental groups were set up: Wild-type control group(WT group), wild-type stimulation group(WT+LPS group), knockout control group (KO group), knockout stimulation group (KO+LPS group), rescue group (KO+LPS+lincRNA-EPS group). In WT+LPS group, KO+LPS group and KO+LPS+lincRNA-EPS group, mPDLCs were stimulated by LPS of 100 ng/mL for 6 hours when WT group and KO group were left untreated. And KO+LPS+lincRNA-EPS group was transfected with lincRNA-EPS overexpressed plasmid in advance. The mRNA levels of inflammatory cytokines including cxcl10, cxcl12, cxcl14, IL-1α and IL-6 were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR). Protein levels of inflammatory cytokines including cxcl10、IL-1α and IL-6 in each group were detected by enzyme-linked immunosorbent assay(ELISA). Results: The C57BL/6J mouse model with lincRNA-EPS knockout was successfully established. MPDLCs were successfully transfected by nanoparticles carrying lincRNA-EPS overexpressed plasmid with a transfection efficiency close to 20%. Without LPS stimulation, the expression of inflammatory cytokines in KO group was low and there was no difference with WT group(P>0.05). Deletion of lincRNA-EPS gene would not cause changes in basal expression of inflammatory cytokines in mPDLCs. After 6 hours of LPS stimulation, the expressions of inflammatory cytokines in KO+LPS group and WT+LPS group were both up-regulated compared with the respective control groups(P<0.05), and KO+LPS group was up-regulated more significantly than WT+LPS group(P<0.05), which indicate that mPDLCs lacking lincRNA-EPS are more likely to trigger strong inflammatory cytokines expressions when stimulated by LPS. Compared with KO+LPS group, the expression of inflammatory cytokines was down-regulated in KO+LPS+lincRNA-EPS group(P<0.05). The transfer of exogenous lincRNA-EPS suppressed the expression of inflammatory cytokines to a certain extent. Conclusion: LincRNA-EPS negatively regulates the expression of inflammatory cytokines in LPS-induced mPDLCs.

Key words: long intergenic noncoding RNA, lipopolysaccharides, periodontitis, mouse periodontal ligament cells

中图分类号: 

• R782