本文選題：Rab5　切入點(diǎn)：Rab7　出處：《南華大學(xué)》2011年碩士論文

【摘要】：目的:以弓形蟲(chóng)PRU-T2株感染巨噬細(xì)胞建立體外感染模型,免疫熒光技術(shù)檢測(cè)含蟲(chóng)空泡膜上介導(dǎo)其與溶酶體融合的三種蛋白(Rab5、Rab7、EEA-1)的動(dòng)態(tài)變化。研究弓形蟲(chóng)抑制含蟲(chóng)空泡與溶酶體融合的機(jī)制,為闡明弓形蟲(chóng)免疫逃逸的機(jī)制及合理設(shè)計(jì)其疫苗提供實(shí)驗(yàn)依據(jù)。 方法: 1建立體外弓形蟲(chóng)感染細(xì)胞模型分別培養(yǎng)小鼠腹腔巨噬細(xì)胞、小鼠脾臟巨噬細(xì)胞、小鼠肺泡巨噬細(xì)胞、RAW264.7鼠傳代巨噬細(xì)胞,用PRU-T2弓形蟲(chóng)感染細(xì)胞(以滅活的PRU-T2弓形蟲(chóng)為陰性對(duì)照),分別于30 min、60 min、90 min及120 min取出蓋玻片,固定,瑞氏染色。比較弓形蟲(chóng)入侵以上4種細(xì)胞形成含蟲(chóng)空泡的時(shí)間與數(shù)量。選擇合適的細(xì)胞用于下一步研究。 2間接免疫熒光技術(shù)分別檢測(cè)Rab5、Rab7、EEA-1在含蟲(chóng)空泡膜上的動(dòng)態(tài)分布取對(duì)數(shù)生長(zhǎng)期細(xì)胞消化吹散;將細(xì)胞鋪于預(yù)置有蓋玻片的24孔板中,待細(xì)胞爬片后將弓形蟲(chóng)加入到孔中感染巨噬細(xì)胞;取出蓋玻片,用4%多聚甲醛固定10-30min;用0.5% Triton-100處理10-20min;加入2%胎牛白蛋白,37℃溫箱中孵育1h封閉;加入適當(dāng)稀釋比的一抗工作液,4℃過(guò)夜;洗滌,加二抗,37℃溫箱中孵育1h;洗滌,晾干,攝像。 結(jié)果: 1比較了PRU-T2弓形蟲(chóng)感染小鼠腹腔巨噬細(xì)胞、小鼠脾臟巨噬細(xì)胞、小鼠肺泡腔巨噬細(xì)胞、RAW264.7細(xì)胞后對(duì)細(xì)胞狀態(tài)的影響及含蟲(chóng)空泡形成的時(shí)間與數(shù)量。從接種鼠腹腔取出4℃放置1-2天的蟲(chóng)體,分別感染小鼠腹腔巨噬細(xì)胞、小鼠脾臟巨噬細(xì)胞、小鼠肺泡腔巨噬細(xì)胞、RAW264.7細(xì)胞,于30min計(jì)數(shù)50個(gè)細(xì)胞中含蟲(chóng)泡的數(shù)量分別是50、10-50、50-80、50-100。 2 Rab5在含蟲(chóng)空泡膜上持續(xù)存在,在熱滅活弓形蟲(chóng)吞噬體上隨著時(shí)間的推移,含量下降直至消失;在含蟲(chóng)空泡膜上檢測(cè)不到Rab7,在熱滅活弓形蟲(chóng)吞噬體膜上Rab7含量升高; EEA1(早內(nèi)體自身抗原1)在含蟲(chóng)空泡膜上無(wú),在熱滅活弓形蟲(chóng)吞噬體膜上存在。 結(jié)論: 1弓形蟲(chóng)侵入RAW264.7細(xì)胞形成含蟲(chóng)空泡的時(shí)間相對(duì)早、數(shù)量相對(duì)多。且RAW264.7細(xì)胞容易培養(yǎng),所以適合下一步研究。 2 Rab5、Rab7、EEA-1三種蛋白在含蟲(chóng)空泡膜上的動(dòng)態(tài)變化提示弓形蟲(chóng)可能通過(guò)保留Rab5、排除Rab7而維持含蟲(chóng)空泡的早內(nèi)體性質(zhì),并通過(guò)阻止EEA-1在含蟲(chóng)空泡上的出現(xiàn)抑制含蟲(chóng)空泡與溶酶體的融合,從而達(dá)到免疫逃逸的目的。
[Abstract]:Aim: to establish an in vitro infection model of macrophages infected with PRU-T2 strain of Toxoplasma gondii, and to detect the dynamic changes of three proteins, Rab5, Rab7, EEA-1, mediated by cytosomal fusion on the vacuolar membrane of Toxoplasma gondii.To study the mechanism of Toxoplasma gondii inhibiting the fusion of vacuoles and lysosomes in order to elucidate the mechanism of immune escape of Toxoplasma gondii and to design its vaccine reasonably.Methods:1 the mouse peritoneal macrophages, mouse spleen macrophages and mouse alveolar macrophages (RAW264.7) were cultured with Toxoplasma gondii infection cell model in vitro.PRU-T2 Toxoplasma gondii infected cells (inactivated PRU-T2 Toxoplasma gondii as negative control) were removed from the glass slides at 30 min, 60 min, 90 min and 120 min, respectively.To compare the time and quantity of invading the above four cells of Toxoplasma gondii to form vacuoles.Select the appropriate cells for further research.2 indirect immunofluorescence technique was used to detect the dynamic distribution of Rab5, Rab7 and EEA-1 on the vacuolar membrane. The cells were digested and dispersed during logarithmic growth, and the cells were placed in a 24 well plate with a cover glass plate.After cell climbing, Toxoplasma gondii was added to the hole to infect macrophages, the cover glass was taken out and fixed with 4% paraformaldehyde for 10-30 min, treated with 0.5% Triton-100 for 10-20 min, and incubated in 37 鈩，

本文編號(hào)：1703281