刚地弓形虫RH株速殖子在鼠星形胶质细胞体外培养的实验观察

发布时间：2018-05-22 16:34

本文选题：星形胶质细胞 + 弓形虫　；参考：《苏州大学》2010年硕士论文

【摘要】： 目的弓形虫感染可引起多种组织器官的病变,其中危害最为严重的是弓形虫穿越血脑屏障入侵人脑引起的弓形虫脑炎。本实验建立刚地弓形虫RH株速殖子体外感染SD大鼠脑星形胶质细胞的模型,研究自噬现象在弓形虫速殖子入侵星形胶质细胞过程中的作用,为探讨弓形虫速殖子感染脑非特异免疫细胞星形胶质细胞的可能的机制提供基础。方法1.原代培养新生SD大鼠大脑皮质混合胶质细胞,经振荡的方法,得到纯化的星形胶质细胞,经免疫组化鉴定细胞纯度。 2.复苏冻存的弓形虫速殖子,腹腔感染昆明小鼠,待到小鼠出现感染症状,抽取腹腔液,观察弓形虫速殖子毒力情况,直到接种三代,收集弓形虫速殖子。 3.待星形胶质细胞传到第三代时,将其接种于预先放有玻片的培养皿中,等到细胞长满培养皿的80%,弓形虫RH株速殖子与星形胶质细胞共培养,0.5～72h,Giemsa染色,观察速殖子的增殖情况。 4.将共培养的弓形虫速殖子与星形胶质细胞,放在活细胞工作站下,观察RH速殖子与星形胶质细胞的作用方式。 5.MDC染色,荧光显微镜下观察0～48h内,共培养的星形胶质细胞内自噬囊泡的变化。结果(1)星形胶质细胞经恒温振荡分离,传至第三代,此时细胞成熟,状态稳定。并经GFAP抗体对星形胶质细胞鉴定,纯度为95%以上。 (2)速殖子可在昆明小鼠体内传代保种,每2～3天传代1次。传至第三代时,弓形虫速殖子毒力完好。 (3)弓形虫速殖子与星形胶质细胞培养2h,有虫体开始进入细胞;8h后进入细胞虫体增多明显;24h绝大多数虫体已经进入细胞;到48h,大多数星形胶质细胞内可以看到虫体形成的假包囊,包囊最初形成时虫体呈菊花状排列,然后以二分裂的方式增殖,逐渐形成成熟的包囊,等待突破细胞膜的时;72h大部分细胞已被虫体涨破,大量虫体从细胞中释放,开始新的感染--假包囊--增殖--破膜的过程。经过这一周期,虫体可增殖10～20倍。 (4)活细胞工作站下,观察到首先弓形虫速殖子以其侧面与星形胶质细胞膜接触,然后再以顶部钻入,弓形虫速殖子是主动入侵鼠星形胶质细胞。 (5)MDC染色,弓形虫速殖子与星形胶质细胞共培养1h,细胞内开始出现点状荧光颗粒;2h、4h、8h,细胞内荧光颗粒逐渐增加,以8h最为显著;在12h时,细胞内荧光颗粒明显减少,伴随细胞出现损伤,但形态尚完整;虫体感染细胞24h和48h时,细胞内荧光颗粒几乎消失,同时细胞被破坏。结论建立了弓形虫RH速殖子体外感染鼠星形胶质细胞的模型;弓形虫速殖子体外感染鼠星形胶质细胞的周期是72h,其体外入侵星形胶质细胞的方式是主动进攻;初步确认自噬在弓形虫速殖子体外感染星形胶质细胞的过程中是受到抑制的。
[Abstract]:Objective Toxoplasma gondii infection can cause a variety of pathological changes in tissues and organs, among which the most serious one is toxoplasma encephalitis caused by Toxoplasma gondii crossing the blood-brain barrier and invading the human brain. In this study, we established a model of Toxoplasma gondii RH strain tachyzoites infecting rat brain astrocytes in vitro, and studied the role of autophagy in the process of Toxoplasma gondii tachyzoites invading astrocytes. To explore the possible mechanism of Toxoplasma gondii tachyzoites infection of brain non-specific immune cells astrocytes. Method 1. Primary cultured mixed glial cells in the cerebral cortex of neonatal SD rats were obtained by oscillating method. The purity of the purified astrocytes was identified by immunohistochemistry. 2. Resuscitated frozen Toxoplasma gondii Toxoplasma gondii Tachyzoites (Toxoplasma gondii) Toxoplasma gondii Toxoplasma gondii Tachyzoites were collected after inoculation for three generations. 3. When astrocytes were transferred to the third generation, they were inoculated in a culture dish with a glass plate. When the cells grew up to 80% of the culture dish, Toxoplasma gondii RH strain Toxoplasma gondii RH strain tachyzoites were co-cultured with astrocytes for 0.572 h and Giemsa staining was used to observe the proliferation of tachyzoites. 4. The co-cultured Toxoplasma gondii tachyzoites and astrocytes were placed under the living cell workstation to observe the interaction between RH tachyzoites and astrocytes. The changes of autophagic vesicles in co-cultured astrocytes were observed by 5.MDC staining and fluorescence microscope within 48 h. Results 1) astrocytes were isolated by isothermal oscillation and passed to the third generation. The cells were mature and stable. The purity of astrocytes was over 95% by GFAP antibody. Tachyzoites can be subcultured and preserved in Kunming mice once every 2 days. At the third generation, Toxoplasma gondii Toxoplasma tachyzoites have good virulence. (3) when Toxoplasma gondii Tachyzoites and astrocytes were cultured for 2 h, there was a marked increase in the number of parasites entering the cells for 8 h, and most of the parasites had entered the cells at 24 h, and at 48 h, the pseudocysts formed by the parasites could be seen in most astrocytes. The cysts were arranged in chrysanthemum form at first, then proliferated in a dimitotic manner, and gradually formed mature cysts. After 72 hours, most of the cells had been broken by the parasites, and a large number of them were released from the cells. Start a new process of infection-pseudocysts-proliferation-rupture of the membrane. After this cycle, the body can multiply by 10 ~ 20 times. Under the living cell workstation, it was observed that Toxoplasma gondii tachyzoites were in contact with astrocytes at the side and then drilled in at the top. Toxoplasma gondii tachyzoites were active invading astrocytes. When Toxoplasma gondii Tachyzoites and astrocytes were co-cultured for 1 h, the dot fluorescent particles began to appear in the cells for 4 h or 8 h, and the intracellular fluorescent particles increased gradually, especially at 8 h, and decreased significantly at 12 h. At 24 h and 48 h after infection, the fluorescent particles almost disappeared and the cells were destroyed. Conclusion the model of rat astrocytes infected with Toxoplasma gondii RH tachyzoites in vitro was established, and the cycle of Toxoplasma gondii Tachyzoites infection in vitro was 72 h, and the way of invading astrocytes in vitro was active attack. It was preliminarily confirmed that autophagy was inhibited during the infection of Toxoplasma gondii tachyzoites in vitro.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R382.5

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