线粒体融合蛋白2基因干扰与过表达对布鲁氏菌诱导巨噬细胞凋亡的影响

Effects of Mitochondrial Fusion Protein 2 Gene Interference and Overexpression on Macrophage Apoptosis Induced by Brucella

【目的】试验旨在探究线粒体融合蛋白2(MFN2)基因干扰与过表达对布鲁氏菌诱导小鼠巨噬细胞凋亡的影响。【方法】利用RNA干扰技术设计3条特异性针对MFN2基因的siRNA序列(siMFN2-450、siMFN2-1661、siMFN2-2275)和1条阴性干扰序列(siMFN2-Negative);利用空质粒pcDNA3.1-EGFP通过过表达技术构建1个重组质粒pcDNA3.1-EGFP-MFN2。双酶切法鉴定过表达重组质粒pcDNA3.1-EGFP-MFN2,通过实时荧光定量PCR和Western blotting检测筛选最佳干扰序列并鉴定重组质粒的过表达效率,使用筛选出的最佳干扰序列和鉴定过表达后的重组质粒分别构建MFN2基因干扰及过表达的转染巨噬细胞模型;用牛种布鲁氏菌疫苗株A19侵染巨噬细胞,按照细菌数∶细胞个数=100∶1的比例侵染24 h,通过实时荧光定量PCR和Western blotting检测B淋巴细胞瘤-2(Bcl-2)相关X蛋白(BAX)的表达情况,用流式细胞术检测细胞凋亡情况。【结果】双酶切结果显示,pcDNA3.1-EGFP-MFN2过表达重组质粒构建成功;实时荧光定量PCR和Western blotting结果显示,siMFN2-1661组MFN2的表达极显著降低(P<0.01),pcDNA3.1-EGFP-MFN2组MFN2的表达极显著增加(P<0.01),成功构建干扰及过表达MFN2基因的转染巨噬细胞模型;布鲁氏菌侵染干扰MFN2基因表达的巨噬细胞后,BAX蛋白表达水平显著高于siMFN2-Negative组(P<0.05),BAX转录水平和细胞凋亡率极显著高于siMFN2-Negative组(P<0.01);布鲁氏菌侵染过表达MFN2基因的巨噬细胞后,BAX的转录及蛋白表达水平均显著低于pcDNA3.1-EGFP组(P<0.05),细胞凋亡率极显著低于pcDNA3.1-EGFP组(P<0.01)。【结论】本试验结果表明,干扰MFN2基因的表达会增强布鲁氏菌诱导细胞凋亡的能力,而过表达MFN2基因则会抑制布鲁氏菌诱导细胞凋亡的能力,结果可为研究MFN2基因的生物学功能提供参考,为进一步解析布鲁氏菌的致病机制提供理论基础。

【Objective】The purpose of this experiment was to investigate the effect of mitochondrial fusion protein 2(MFN2)gene interference and overexpression on mouse macrophages apoptosis induced by Brucella.【Method】Three siRNA sequences,siMFN2-450,siMFN2-1661,siMFN2-2275 and one negative interference sequence siMFN2-Negative specifically targeting MFN2 gene were designed by RNA interference techniques.A recombinant plasmid pcDNA3.1-EGFP-MFN2 was constructed by overexpression technique using empty plasmid pcDNA3.1-EGFP.The overexpression of recombinant plasmid pcDNA3.1-EGFP-MFN2 was identified by double enzyme digestion.The best interference sequence was screened and the overexpression efficiency of the recombinant plasmid was identified by Real-time quantitative PCR and Western blotting.The optimal interference sequence and the overexpressed recombinant plasmid were used to construct the macrophage model of MFN2 gene interference and overexpression,respectively.Macrophages were infected with Brucella bovis vaccine strain A19 for 24 hours according to the ratio of bacterial number to cell number(100∶1).The expression of Bcl-2 related X protein(BAX)was detected by Real-time quantitative PCR and Western blotting,and cell apoptosis was detected by flow cytometry.【Result】The results of double enzyme digestion showed that the overexpression recombinant plasmid pcDNA3.1-EGFP-MFN2 was successfully constructed.Real-time quantitative PCR and Western blotting results showed that siMFN2-1661 could extremely significantly inhibit the expression of MFN2(P<0.01),and pcDNA3.1-EGFP-MFN2 could extremely significantly enhance the expression of MFN2(P<0.01).The transfected macrophage model with interference and overexpression of MFN2 gene was successfully constructed.After macrophages interfering with MFN2 gene expression were infected by Brucella,the protein expression levels of BAX was significantly higher than that in siMFN2-Negative group(P<0.05),transcription levels and the apoptosis rate of BAX were extremely significantly higher than that in siMFN2-Negative group(P<0.01).After macrophages overexpressing MFN2 gene were infected by Brucella,the transcription and protein expression of BAX were significantly lower than those in pcDNA3.1-EGFP group(P<0.05),and the apoptosis rate was extremely significantly lower than that in pcDNA3.1-EGFP group(P<0.01).【Conclusion】The study showed that MFN2 gene interference would enhance the ability of Brucella to induce cell apoptosis,while MFN2 gene overexpression would inhibit the ability of Brucella to induce cell apoptosis.The results could provide a reference for studying biological function of MFN2 gene and provide a theoretical basis for further analysis the pathogenic mechanism of Brucella.