摘要
为了探讨E3泛素连接酶Nrdp1、SOCS-1基因在布鲁氏菌侵染巨噬细胞过程中对细胞凋亡的影响,构建Nrdp1、SOCS-1基因的干扰和过表达细胞模型(简称为pLL3.7-N1、pLL3.7-S1和pLEX-Nrdp1、pLEX-SOCS-1);羊种布鲁氏菌16M(简称16M)侵染正常细胞RAW264.7及pLL3.7-N1、pLEX-Nrdp1、pLL3.7-S1、pLEX-SOCS-1组细胞,qRT-PCR检测Bax、Bcl-2、TNF-α的mRNA表达量,并通过流式细胞仪术检测细胞凋亡率。结果显示,试验成功构建并筛选出干扰和过表达Nrdp1、SOCS-1效果最好的pLL3.7-N1、pLEX-Nrdp1、pLL3.7-S1、pLEXSOCS-1细胞模型;16M侵染各组细胞,与对照组相比,在不同的时间段pLL3.7-N1、pLL3.7-S1、pLEX-Nrdp1、pLEX-SOCS-1组细胞的Bcl-2和Bax的mRNA表达量差异显著(P<0.05);pLL3.7-S1组细胞的TNF-αmRNA显著降低(P<0.05),而pLEX-SOCS-1组显著升高(P<0.05);pLEX-Nrdp1、pLEX-SOCS-1组细胞凋亡率显著升高(P<0.05),而pLL3.7-S1组显著降低(P<0.05)。研究结果表明,Nrdp1、SOCS-1基因与16M诱导的细胞凋亡密切相关,为16M胞内寄生机制的研究奠定了基础。
In order to investigate the effect of E3 ubiquitin ligase Nrdp1 and SOCS-1genes on apoptosis during Brucellainfection macrophages.The cell models of interference and over expression of Nrdp1 and SOCS-1genes（pLL3.7-N1,pLL3.7-S1 and pLEX-Nrdp1,pLEX-SOCS-1）were constructed.Normal RAW264.7cell,pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1 and pLEXSOCS-1group cells were infected with Brucella melitensis 16M（referred to 16M）,the expression of Bax,Bcl-2,TNF-αgenes were detected by qRT-PCR and apoptosis rate was detected by flow cytometry.pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1,pLEX-SOCS-1cell model of the works best of interference and overexpression Nrdp1,SOCS-1were successfully constructed and screened.After16 Minfecting each group cells,compared with the control group,the expression of Bcl-2and Bax mRNA in pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1 and pLEX-SOCS-1groups were significantly different in different time periods（P〈0.05）.The expression of TNF-αmRNA in pLL3.7-S1 group was significantly lower（P〈0.05）,while the pLEX-SOCS-1group was significantly higher（P〈0.05）.Apoptosis rates in pLEX-Nrdp1,pLEX-SOCS-1groups were significantly higher（P〈0.05）,while pLL3.7-S1 group was lower（P〈0.05）.The results showed that the Nrdp1 and SOCS-1genes were closely related to apoptosis with 16 Minduction,the research laid the foundation for the study of 16 Mintracellular parasitism mechanisms.
出处
《中国畜牧兽医》
CAS
北大核心
2016年第6期1393-1404,共12页
China Animal Husbandry & Veterinary Medicine
基金
国家科技合作项目(2013DFA32380)
国家科技支撑计划子课题(2013BAI05B05)
国家自然科学基金(1572491
31502067
31260596)
自治区研究生教育创新计划科研创新项目(XJGRI2015040)
