本文選題：布魯氏菌 + 巨噬細(xì)胞　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：布魯氏菌病(Brucellosis)簡(jiǎn)稱(chēng)布病,是由布魯氏菌屬(Brucella)的布魯氏菌侵入機(jī)體,引起的一種慢性人畜共患傳染病病。該病在世界范圍內(nèi)廣泛流行,嚴(yán)重危害公共衛(wèi)生安全,并造成畜牧業(yè)經(jīng)濟(jì)巨大損失。巨噬細(xì)胞是布魯氏菌感染的首要宿主細(xì)胞,布魯氏菌感染巨噬細(xì)胞后可以激發(fā)不依賴(lài)Atg5(Autophagy related gene 5)的自噬,且逃避其溶酶體的降解。自噬相關(guān)蛋白Atg5和膜融合相關(guān)蛋白Tecpr1(Tectonin domain-containing protein)的結(jié)構(gòu)域AIR(Atg12-Atg5-interacting region)在促進(jìn)自噬體與溶酶體的融合中有著重要作用,但在布魯氏菌誘導(dǎo)的自噬中這兩種蛋白的作用尚不清楚。目的:探究Atg5和AIR結(jié)構(gòu)域?qū)ρ蚍N布魯氏菌16M感染小鼠巨噬細(xì)胞RAW264.7引起的非典型自噬中自噬體與溶酶體的融合及對(duì)16M胞內(nèi)生存繁殖能力的影響。方法:通過(guò)分子生物學(xué)手段分別構(gòu)建表達(dá)Atg5和沉默AIR的慢病毒載體;重組慢病毒質(zhì)粒、包裝質(zhì)粒、包膜質(zhì)粒三質(zhì)粒共轉(zhuǎn)染到293T細(xì)胞包裝慢病毒,轉(zhuǎn)染48h后通過(guò)觀(guān)察綠色熒光鑒定病毒包裝是否成功;將包裝好的病毒進(jìn)一步感染RAW264.7,QRT-PCR檢測(cè)Atg5和AIR的表達(dá)。用羊種布魯氏菌16M菌株分別感染表達(dá)Atg5和沉默AIR基因的RAW264.7細(xì)胞,并以感染正常RAW264.7細(xì)胞為空白對(duì)照;感染4 h、12 h、24 h、48 h后,通過(guò)CFU計(jì)數(shù)實(shí)驗(yàn)檢測(cè)胞內(nèi)存活的布魯氏菌數(shù)量,并通過(guò)激光共聚焦檢測(cè)布魯氏菌感染24 h后細(xì)胞內(nèi)自噬體與溶酶體融合的情況。QRT-PCR檢測(cè)布魯氏菌感染24 h后自噬相關(guān)蛋白LC3Ⅰ/Ⅱ、p62在m RNA水平上的表達(dá)情況,Western blot檢測(cè)自噬相關(guān)蛋白LC3Ⅰ/Ⅱ、p62的蛋白表達(dá)量。結(jié)果:限制性?xún)?nèi)切酶雙酶切鑒定以及DNA測(cè)序表明Atg5和AIR重組慢病毒質(zhì)粒構(gòu)建成功;慢病毒包裝實(shí)驗(yàn)發(fā)現(xiàn)重組病毒轉(zhuǎn)染細(xì)胞后可觀(guān)察到細(xì)胞內(nèi)有許多綠色熒光點(diǎn)。QRT-PCR實(shí)驗(yàn)結(jié)果表明表達(dá)Atg5和沉默AIR慢病毒載體轉(zhuǎn)染成功。布魯氏菌感染過(guò)表達(dá)Atg5和沉默AIR的RAW264.7后,激光共聚焦結(jié)果顯示實(shí)驗(yàn)組的自噬體和溶酶體融合情況明顯高于對(duì)照組(P0.05)。QRT-PCR和Western blot實(shí)驗(yàn)表明,與對(duì)照組相比,布魯氏菌感染過(guò)表達(dá)Atg5的RAW264.7后,自噬相關(guān)蛋白LC3Ⅰ/Ⅱ、p62的表達(dá)量下降(P0.05)。細(xì)菌胞內(nèi)生存CFU實(shí)驗(yàn)表明,與對(duì)照組相比,布魯氏菌16M感染過(guò)表達(dá)Atg5和沉默AIR基因的巨噬細(xì)胞后,胞內(nèi)細(xì)菌數(shù)明顯降低(P0.05)。結(jié)論:在布魯氏菌16M感染RAW264.7細(xì)胞誘發(fā)的非典型自噬中,過(guò)表達(dá)Atg5和沉默AIR會(huì)通過(guò)促進(jìn)自噬體與溶酶體的融合,在一定程度上降低布魯氏菌在宿主細(xì)胞內(nèi)的生存能力。這為我們進(jìn)一步研究布魯氏菌通過(guò)哪種途徑阻滯自噬體與溶酶體的融合從而在宿主細(xì)胞內(nèi)持續(xù)生存的相關(guān)機(jī)制奠定了實(shí)驗(yàn)基礎(chǔ)。
[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，

本文編號(hào)：2079115