弓形虫抑制含虫空泡与溶酶体融合机制的初步研究

发布时间：2018-04-03 02:39

  本文选题：Rab5　切入点：Rab7　出处：《南华大学》2011年硕士论文

【摘要】：目的:以弓形虫PRU-T2株感染巨噬细胞建立体外感染模型,免疫荧光技术检测含虫空泡膜上介导其与溶酶体融合的三种蛋白(Rab5、Rab7、EEA-1)的动态变化。研究弓形虫抑制含虫空泡与溶酶体融合的机制,为阐明弓形虫免疫逃逸的机制及合理设计其疫苗提供实验依据。 方法: 1建立体外弓形虫感染细胞模型分别培养小鼠腹腔巨噬细胞、小鼠脾脏巨噬细胞、小鼠肺泡巨噬细胞、RAW264.7鼠传代巨噬细胞,用PRU-T2弓形虫感染细胞(以灭活的PRU-T2弓形虫为阴性对照),分别于30 min、60 min、90 min及120 min取出盖玻片,固定,瑞氏染色。比较弓形虫入侵以上4种细胞形成含虫空泡的时间与数量。选择合适的细胞用于下一步研究。 2间接免疫荧光技术分别检测Rab5、Rab7、EEA-1在含虫空泡膜上的动态分布取对数生长期细胞消化吹散;将细胞铺于预置有盖玻片的24孔板中,待细胞爬片后将弓形虫加入到孔中感染巨噬细胞;取出盖玻片,用4%多聚甲醛固定10-30min;用0.5% Triton-100处理10-20min;加入2%胎牛白蛋白,37℃温箱中孵育1h封闭;加入适当稀释比的一抗工作液,4℃过夜;洗涤,加二抗,37℃温箱中孵育1h;洗涤,晾干,摄像。 结果: 1比较了PRU-T2弓形虫感染小鼠腹腔巨噬细胞、小鼠脾脏巨噬细胞、小鼠肺泡腔巨噬细胞、RAW264.7细胞后对细胞状态的影响及含虫空泡形成的时间与数量。从接种鼠腹腔取出4℃放置1-2天的虫体,分别感染小鼠腹腔巨噬细胞、小鼠脾脏巨噬细胞、小鼠肺泡腔巨噬细胞、RAW264.7细胞,于30min计数50个细胞中含虫泡的数量分别是50、10-50、50-80、50-100。 2 Rab5在含虫空泡膜上持续存在,在热灭活弓形虫吞噬体上随着时间的推移,含量下降直至消失;在含虫空泡膜上检测不到Rab7,在热灭活弓形虫吞噬体膜上Rab7含量升高; EEA1(早内体自身抗原1)在含虫空泡膜上无,在热灭活弓形虫吞噬体膜上存在。 结论: 1弓形虫侵入RAW264.7细胞形成含虫空泡的时间相对早、数量相对多。且RAW264.7细胞容易培养,所以适合下一步研究。 2 Rab5、Rab7、EEA-1三种蛋白在含虫空泡膜上的动态变化提示弓形虫可能通过保留Rab5、排除Rab7而维持含虫空泡的早内体性质,并通过阻止EEA-1在含虫空泡上的出现抑制含虫空泡与溶酶体的融合,从而达到免疫逃逸的目的。
[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，

本文编号：1703281