摘要
目的 通过干扰小鼠巨噬细胞Shc1基因,探究Shc1对脂多糖LPS刺激巨噬细胞极化的作用。方法 分离C57BL/6J成年小鼠(6~8周)的巨噬细胞,加入LPS刺激24 h后,提取细胞RNA反转录为cDNA,利用siRNA技术敲低Shc1基因(si-Shc1),和对照组(Si-NC)同时处理48 h后检测Shc1的mRNA和蛋白的敲低水平,利用RT-qPCR分别检测促炎因子、转录因子和抑炎因子的mRNA水平变化;在Si-NC和si-Shc1组中使用脂多糖LPS刺激24 h后,利用RT-qPCR分别检测促炎因子、转录因子和抑炎因子的mRNA水平变化。结果 LPS刺激可以促进M1型巨噬细胞分泌促炎因子(P<0.000 1),且增强转录因子的RNA水平(P<0.000 1);与si-NC组相比,si-Shc1会增强细胞中促炎因子TNF-α的RNA水平(P<0.001),降低IL-1β、IL-6和iNOS的RNA水平(P<0.001);敲低Shc1的细胞加入LPS刺激后,促炎因子以及转录因子的RNA水平与未处理细胞组相当。结论 Shc1参与调控促炎因子的表达,但不参与脂多糖LPS对M1型巨噬细胞的极化作用。
Objective To explore Shc1 for lipopolysaccharide LPS activated macrophages polarization by interfering with Shc1 gene in mice macrophages.Methods We isolated macrophages from adult C57BL/6J mice(6-8 weeks old),stimulated them with LPS for 24 h,extracted cellular RNA and reverse transcribed it into cDNA.RT-qPCR was used to detect changes in the mRNA levels of pro-inflammatory cytokines,transcription factors,and anti-inflammatory cytokines.Macrophages were isolated from adult mice(6-8 weeks old),and Shc1 gene was knocked down using siRNA technology(si-Shc1)along with a control group(Si-NC)treated for 48 h.The mRNA and protein levels of Shc1 were detected,and RT-qPCR was used to detect changes in the mRNA levels of pro-inflammatory cytokines,transcription factors,and anti-inflammatory cytokines.In both Si-NC and si-Shc1 groups,LPS was used to stimulate cells for 24 h,and RT-qPCR was used to detect changes in the mRNA levels of pro-inflammatory cytokines,transcription factors,and anti-inflammatory cytokines.Results LPS can promote the proinflammatory factor secretion of macrophages(P<0.0001),and enhance the RNA level of transcription factors(P<0.0001);compared with si-NC group,si-Shc1 group increased the RNA level of proinflammatory factor TNF-α(P<0.001),and reduced RNA level of IL-1β,IL–6 and iNOS(P<0.001).When si-Shc1 cells were added LPS,RNA levels of pro-inflammatory factors as well as transcription factors were comparable to those of untreated cell groups.Conclusion Shc1 is involved in regulating the expression of pro-inflammatory factors,but not involved in the polarization of lipopolysaccharide LPS on M1 macrophages.
作者
胡苗清
黄成珍
陈厚早
聂宇
廉虹
HU Miaoqing;HUANG Chengzhen;CHEN Houzao;NIE Yu;LIAN Hong(State Key Laboratory of Cardiovascular Disease,Fuwai Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100037,China;Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100037;Beijing Key Laboratory of Preclinical Research and Evaluation for Cardiovascular Implant Materials,Animal Experimental Centre,Fuwai Hospital,National Centre for Cardiovascular Disease,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100037,China)
出处
《中国分子心脏病学杂志》
CAS
2023年第4期5548-5553,共6页
Molecular Cardiology of China
基金
中国医学科学院阜外医院心血管疾病国家重点实验室重点项目(开放运行2022ZD-06)。
