本文選題：星形膠質(zhì)細(xì)胞 + 弓形蟲(chóng)�。� 參考：《蘇州大學(xué)》2010年碩士論文

【摘要】： 目的弓形蟲(chóng)感染可引起多種組織器官的病變,其中危害最為嚴(yán)重的是弓形蟲(chóng)穿越血腦屏障入侵人腦引起的弓形蟲(chóng)腦炎。本實(shí)驗(yàn)建立剛地弓形蟲(chóng)RH株速殖子體外感染SD大鼠腦星形膠質(zhì)細(xì)胞的模型,研究自噬現(xiàn)象在弓形蟲(chóng)速殖子入侵星形膠質(zhì)細(xì)胞過(guò)程中的作用,為探討弓形蟲(chóng)速殖子感染腦非特異免疫細(xì)胞星形膠質(zhì)細(xì)胞的可能的機(jī)制提供基礎(chǔ)。 方法1.原代培養(yǎng)新生SD大鼠大腦皮質(zhì)混合膠質(zhì)細(xì)胞,經(jīng)振蕩的方法,得到純化的星形膠質(zhì)細(xì)胞,經(jīng)免疫組化鑒定細(xì)胞純度。 2.復(fù)蘇凍存的弓形蟲(chóng)速殖子,腹腔感染昆明小鼠,待到小鼠出現(xiàn)感染癥狀,抽取腹腔液,觀察弓形蟲(chóng)速殖子毒力情況,直到接種三代,收集弓形蟲(chóng)速殖子。 3.待星形膠質(zhì)細(xì)胞傳到第三代時(shí),將其接種于預(yù)先放有玻片的培養(yǎng)皿中,等到細(xì)胞長(zhǎng)滿培養(yǎng)皿的80%,弓形蟲(chóng)RH株速殖子與星形膠質(zhì)細(xì)胞共培養(yǎng),0.5～72h,Giemsa染色,觀察速殖子的增殖情況。 4.將共培養(yǎng)的弓形蟲(chóng)速殖子與星形膠質(zhì)細(xì)胞,放在活細(xì)胞工作站下,觀察RH速殖子與星形膠質(zhì)細(xì)胞的作用方式。 5.MDC染色,熒光顯微鏡下觀察0～48h內(nèi),共培養(yǎng)的星形膠質(zhì)細(xì)胞內(nèi)自噬囊泡的變化。 結(jié)果(1)星形膠質(zhì)細(xì)胞經(jīng)恒溫振蕩分離,傳至第三代,此時(shí)細(xì)胞成熟,狀態(tài)穩(wěn)定。并經(jīng)GFAP抗體對(duì)星形膠質(zhì)細(xì)胞鑒定,純度為95%以上。 (2)速殖子可在昆明小鼠體內(nèi)傳代保種,每2～3天傳代1次。傳至第三代時(shí),弓形蟲(chóng)速殖子毒力完好。 (3)弓形蟲(chóng)速殖子與星形膠質(zhì)細(xì)胞培養(yǎng)2h,有蟲(chóng)體開(kāi)始進(jìn)入細(xì)胞;8h后進(jìn)入細(xì)胞蟲(chóng)體增多明顯;24h絕大多數(shù)蟲(chóng)體已經(jīng)進(jìn)入細(xì)胞;到48h,大多數(shù)星形膠質(zhì)細(xì)胞內(nèi)可以看到蟲(chóng)體形成的假包囊,包囊最初形成時(shí)蟲(chóng)體呈菊花狀排列,然后以二分裂的方式增殖,逐漸形成成熟的包囊,等待突破細(xì)胞膜的時(shí);72h大部分細(xì)胞已被蟲(chóng)體漲破,大量蟲(chóng)體從細(xì)胞中釋放,開(kāi)始新的感染--假包囊--增殖--破膜的過(guò)程。經(jīng)過(guò)這一周期,蟲(chóng)體可增殖10～20倍。 (4)活細(xì)胞工作站下,觀察到首先弓形蟲(chóng)速殖子以其側(cè)面與星形膠質(zhì)細(xì)胞膜接觸,然后再以頂部鉆入,弓形蟲(chóng)速殖子是主動(dòng)入侵鼠星形膠質(zhì)細(xì)胞。 (5)MDC染色,弓形蟲(chóng)速殖子與星形膠質(zhì)細(xì)胞共培養(yǎng)1h,細(xì)胞內(nèi)開(kāi)始出現(xiàn)點(diǎn)狀熒光顆粒;2h、4h、8h,細(xì)胞內(nèi)熒光顆粒逐漸增加,以8h最為顯著;在12h時(shí),細(xì)胞內(nèi)熒光顆粒明顯減少,伴隨細(xì)胞出現(xiàn)損傷,但形態(tài)尚完整;蟲(chóng)體感染細(xì)胞24h和48h時(shí),細(xì)胞內(nèi)熒光顆粒幾乎消失,同時(shí)細(xì)胞被破壞。 結(jié)論建立了弓形蟲(chóng)RH速殖子體外感染鼠星形膠質(zhì)細(xì)胞的模型;弓形蟲(chóng)速殖子體外感染鼠星形膠質(zhì)細(xì)胞的周期是72h,其體外入侵星形膠質(zhì)細(xì)胞的方式是主動(dòng)進(jìn)攻;初步確認(rèn)自噬在弓形蟲(chóng)速殖子體外感染星形膠質(zhì)細(xì)胞的過(guò)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R382.5

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本文編號(hào)：1922825