布鲁氏菌侵染巨噬细胞后对自噬—溶酶体降解途径的影响

发布时间：2018-06-28 19:36

  本文选题：布鲁氏菌 + 巨噬细胞　； 参考：《石河子大学》2015年硕士论文

【摘要】：布鲁氏菌病(Brucellosis)简称布病,是由布鲁氏菌属(Brucella)的布鲁氏菌侵入机体,引起的一种慢性人畜共患传染病病。该病在世界范围内广泛流行,严重危害公共卫生安全,并造成畜牧业经济巨大损失。巨噬细胞是布鲁氏菌感染的首要宿主细胞,布鲁氏菌感染巨噬细胞后可以激发不依赖Atg5(Autophagy related gene 5)的自噬,且逃避其溶酶体的降解。自噬相关蛋白Atg5和膜融合相关蛋白Tecpr1(Tectonin domain-containing protein)的结构域AIR(Atg12-Atg5-interacting region)在促进自噬体与溶酶体的融合中有着重要作用,但在布鲁氏菌诱导的自噬中这两种蛋白的作用尚不清楚。目的:探究Atg5和AIR结构域对羊种布鲁氏菌16M感染小鼠巨噬细胞RAW264.7引起的非典型自噬中自噬体与溶酶体的融合及对16M胞内生存繁殖能力的影响。方法:通过分子生物学手段分别构建表达Atg5和沉默AIR的慢病毒载体;重组慢病毒质粒、包装质粒、包膜质粒三质粒共转染到293T细胞包装慢病毒,转染48h后通过观察绿色荧光鉴定病毒包装是否成功;将包装好的病毒进一步感染RAW264.7,QRT-PCR检测Atg5和AIR的表达。用羊种布鲁氏菌16M菌株分别感染表达Atg5和沉默AIR基因的RAW264.7细胞,并以感染正常RAW264.7细胞为空白对照;感染4 h、12 h、24 h、48 h后,通过CFU计数实验检测胞内存活的布鲁氏菌数量,并通过激光共聚焦检测布鲁氏菌感染24 h后细胞内自噬体与溶酶体融合的情况。QRT-PCR检测布鲁氏菌感染24 h后自噬相关蛋白LC3Ⅰ/Ⅱ、p62在m RNA水平上的表达情况,Western blot检测自噬相关蛋白LC3Ⅰ/Ⅱ、p62的蛋白表达量。结果:限制性内切酶双酶切鉴定以及DNA测序表明Atg5和AIR重组慢病毒质粒构建成功;慢病毒包装实验发现重组病毒转染细胞后可观察到细胞内有许多绿色荧光点。QRT-PCR实验结果表明表达Atg5和沉默AIR慢病毒载体转染成功。布鲁氏菌感染过表达Atg5和沉默AIR的RAW264.7后,激光共聚焦结果显示实验组的自噬体和溶酶体融合情况明显高于对照组(P0.05)。QRT-PCR和Western blot实验表明,与对照组相比,布鲁氏菌感染过表达Atg5的RAW264.7后,自噬相关蛋白LC3Ⅰ/Ⅱ、p62的表达量下降(P0.05)。细菌胞内生存CFU实验表明,与对照组相比,布鲁氏菌16M感染过表达Atg5和沉默AIR基因的巨噬细胞后,胞内细菌数明显降低(P0.05)。结论:在布鲁氏菌16M感染RAW264.7细胞诱发的非典型自噬中,过表达Atg5和沉默AIR会通过促进自噬体与溶酶体的融合,在一定程度上降低布鲁氏菌在宿主细胞内的生存能力。这为我们进一步研究布鲁氏菌通过哪种途径阻滞自噬体与溶酶体的融合从而在宿主细胞内持续生存的相关机制奠定了实验基础。
[Abstract]:Brucellosis is a chronic zoonotic disease caused by Brucella. The disease is widespread in the world, seriously endangering public health and safety, and causing huge loss of animal husbandry economy. Macrophages are the primary host cells of brucella infection. Brucellosis infected macrophages can stimulate autophagy independent of Atg5 (Autophagy related gene 5 and escape the degradation of lysosomes. The domain air (Atg12-Atg5-interacting region) of autophagy associated protein Atg5 and membrane fusion protein Tecpr1 (Tectonin domain-containing protein) plays an important role in promoting the fusion of autophagy and lysosome, but the roles of these two proteins in brucella-induced autophagy are still unclear. Aim: to investigate the effect of Atg5 and air domain on the fusion of autophagy and lysosome in mouse macrophage RAW264.7 induced by 16M sheep brucella and its effect on the viability and reproduction of 16M mouse macrophage. Methods: the lentivirus vectors expressing Atg5 and silencing air were constructed by molecular biology, and the recombinant lentivirus plasmid, the packaging plasmid and the encapsulated plasmid were co-transfected into 293T cells to package the lentivirus. After 48 hours of transfection, green fluorescence was observed to determine whether the virus was successfully packaged, and the virus was further infected with RAW264.7 QRT-PCR to detect the expression of Atg5 and air. RAW264.7 cells expressing Atg5 and silencing air gene were infected with 16M strain of Brucella from sheep, and the normal RAW264.7 cells were infected as blank control, and the number of Brucella that survived in the cell was detected by CFU-counting experiment after infection for 4 h or 12 h or 24 h or 48 h. The fusion of intracellular autophagy and lysosome was detected by confocal laser scanning. QRT-PCR was used to detect the expression of autophagy associated protein LC3 鈪，

本文编号：2079115