本文選題：布魯氏菌 + 巨噬細胞��； 參考：《石河子大學》2015年碩士論文

【摘要】：布魯氏菌病(Brucellosis)簡稱布病,是由布魯氏菌屬(Brucella)的布魯氏菌侵入機體,引起的一種慢性人畜共患傳染病病。該病在世界范圍內廣泛流行,嚴重危害公共衛(wèi)生安全,并造成畜牧業(yè)經濟巨大損失。巨噬細胞是布魯氏菌感染的首要宿主細胞,布魯氏菌感染巨噬細胞后可以激發(fā)不依賴Atg5(Autophagy related gene 5)的自噬,且逃避其溶酶體的降解。自噬相關蛋白Atg5和膜融合相關蛋白Tecpr1(Tectonin domain-containing protein)的結構域AIR(Atg12-Atg5-interacting region)在促進自噬體與溶酶體的融合中有著重要作用,但在布魯氏菌誘導的自噬中這兩種蛋白的作用尚不清楚。目的:探究Atg5和AIR結構域對羊種布魯氏菌16M感染小鼠巨噬細胞RAW264.7引起的非典型自噬中自噬體與溶酶體的融合及對16M胞內生存繁殖能力的影響。方法:通過分子生物學手段分別構建表達Atg5和沉默AIR的慢病毒載體;重組慢病毒質粒、包裝質粒、包膜質粒三質粒共轉染到293T細胞包裝慢病毒,轉染48h后通過觀察綠色熒光鑒定病毒包裝是否成功;將包裝好的病毒進一步感染RAW264.7,QRT-PCR檢測Atg5和AIR的表達。用羊種布魯氏菌16M菌株分別感染表達Atg5和沉默AIR基因的RAW264.7細胞,并以感染正常RAW264.7細胞為空白對照;感染4 h、12 h、24 h、48 h后,通過CFU計數實驗檢測胞內存活的布魯氏菌數量,并通過激光共聚焦檢測布魯氏菌感染24 h后細胞內自噬體與溶酶體融合的情況。QRT-PCR檢測布魯氏菌感染24 h后自噬相關蛋白LC3Ⅰ/Ⅱ、p62在m RNA水平上的表達情況,Western blot檢測自噬相關蛋白LC3Ⅰ/Ⅱ、p62的蛋白表達量。結果:限制性內切酶雙酶切鑒定以及DNA測序表明Atg5和AIR重組慢病毒質粒構建成功;慢病毒包裝實驗發(fā)現(xiàn)重組病毒轉染細胞后可觀察到細胞內有許多綠色熒光點。QRT-PCR實驗結果表明表達Atg5和沉默AIR慢病毒載體轉染成功。布魯氏菌感染過表達Atg5和沉默AIR的RAW264.7后,激光共聚焦結果顯示實驗組的自噬體和溶酶體融合情況明顯高于對照組(P0.05)。QRT-PCR和Western blot實驗表明,與對照組相比,布魯氏菌感染過表達Atg5的RAW264.7后,自噬相關蛋白LC3Ⅰ/Ⅱ、p62的表達量下降(P0.05)。細菌胞內生存CFU實驗表明,與對照組相比,布魯氏菌16M感染過表達Atg5和沉默AIR基因的巨噬細胞后,胞內細菌數明顯降低(P0.05)。結論:在布魯氏菌16M感染RAW264.7細胞誘發(fā)的非典型自噬中,過表達Atg5和沉默AIR會通過促進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胞內的生存能力。這為我們進一步研究布魯氏菌通過哪種途徑阻滯自噬體與溶酶體的融合從而在宿主細胞內持續(xù)生存的相關機制奠定了實驗基礎。
[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：2079115