弓形蟲感染誘導(dǎo)小膠質(zhì)細胞的活化以及米諾環(huán)素的抑制作用
發(fā)布時間：2019-03-24 16:40
【摘要】：背景及目的弓形蟲是一種專性細胞內(nèi)寄生原蟲，能感染包括人在內(nèi)的幾乎所有的恒溫動物。無論是先天性還是獲得性感染，都對人類的健康產(chǎn)生嚴(yán)重的威脅。先天性弓形蟲病主要表現(xiàn)為腦積水、腦鈣化、出生后智力發(fā)育遲緩、癲癇等；獲得性弓形蟲病可表現(xiàn)為頭痛、發(fā)熱、淋巴結(jié)腫大，免疫力低下患者可并發(fā)全身嚴(yán)重弓形蟲病，例如弓形蟲腦炎等。在機體免疫缺陷或功能低下時，腦部的包囊破裂，緩殖子向速殖子轉(zhuǎn)化，弓形蟲隱性感染狀態(tài)被活化，導(dǎo)致致命性弓形蟲腦炎的發(fā)生。弓形蟲不同的基因型與疾病的發(fā)生、發(fā)展以及結(jié)局有關(guān)。在北美和歐洲，主要的弓形蟲基因型為Type I、Type II和Type III,而我國的優(yōu)勢基因型為Chinese1型。小膠質(zhì)細胞，是中樞神經(jīng)系統(tǒng)中主要的固有免疫成分，包括靜息和活化兩種狀態(tài)，在腦部病理損傷時發(fā)揮著重要的作用。在感染、損傷等刺激時，小膠質(zhì)細胞能活化，分化、增殖并分泌大量的神經(jīng)毒性因子，發(fā)揮免疫保護功能，但是小膠質(zhì)細胞過度的活化也會對神經(jīng)元等周圍組織產(chǎn)生損傷。在弓形蟲感染中，對于小膠質(zhì)細胞的影響尚不十分清楚。本文主要研究小膠質(zhì)細胞（BV-2）細胞系在弓形蟲TgCtwh6蟲株（Chinese1基因型，成囊株）感染中的變化，并加入了小膠質(zhì)細胞的抑制劑米諾環(huán)素(minocycline)，以了解其對小膠質(zhì)細胞的抑制作用，進而為弓形蟲腦病的防治提供實驗基礎(chǔ)。 方法在transwell共培養(yǎng)體系中用弓形蟲TgCtwh6蟲株感染BV-2細胞，同時用米諾環(huán)素進行干預(yù)，分為普通培養(yǎng)基對照組、弓形蟲TgCtwh6蟲株感染的代謝物作用組和米諾環(huán)素抑制劑組。在倒置顯微鏡下觀察細胞的形態(tài)；用流式細胞術(shù)檢測細胞凋亡情況；ELISA法檢測細胞上清中IL-1β、IL-6、TNF-α的含量；用Griess法檢測細胞上清中NO的含量；RT-PCR法檢測BV-2細胞IL-1β、IL-6、TNF-α、iNOS mRNA的表達；Western blotting檢測BV-2細胞iNOS蛋白的表達。 結(jié)果與對照組相比，弓形蟲代謝物作用組的小膠質(zhì)細胞胞體變圓，突起減少；流式結(jié)果顯示BV-2細胞感染弓形蟲24h且BV-2細胞與弓形蟲速殖子比例為1:1時出現(xiàn)凋亡，培養(yǎng)48h,BV-2細胞與弓形蟲速殖子比例為1:5時BV-2細胞的凋亡率達35.72%；弓形蟲代謝物作用組的BV-2細胞分泌炎癥因子，其中培養(yǎng)上清中IL-1β、IL-6、TNF-α、NO的含量、BV-2細胞IL-1β、IL-6、TNF-α、iNOS mRNA的表達以及iNOS蛋白的表達均隨著培養(yǎng)時間的延長和弓形蟲速殖子與BV-2細胞比例的提高逐漸增加，米諾環(huán)素能抑制上述炎癥因子的產(chǎn)生。 結(jié)論我國流行的弓形蟲Chinese1基因型TgCtwh6成囊株的代謝物作用的BV-2細胞能分泌IL-1β、IL-6、TNF-α等炎癥因子，，其在對抗弓形蟲的過程中可能發(fā)揮一定的作用，但是對神經(jīng)元細胞也有一定的損傷；米諾環(huán)素可以抑制炎癥因子的產(chǎn)生，提示該藥對弓形蟲腦病具有潛在的治療價值。但是弓形蟲TgCtwh6誘導(dǎo)小膠質(zhì)細胞分泌炎癥因子以及米諾環(huán)素抑制的機制尚待進一步研究。
[Abstract]:Background and objective Toxoplasma gondii is a specific intracellular parasite that infects almost all thermostat animals, including humans. Both congenital and acquired infections pose a serious threat to human health. The main manifestations of congenital toxoplasmosis were hydrocephalus, calcification, postnatal mental retardation, epilepsy and so on. Acquired toxoplasmosis can be characterized by headache fever lymph node enlargement and severe toxoplasmosis in patients with low immunity such as toxoplasmosis encephalitis. In the case of immune deficiency or hypofunction, the cyst of the brain breaks down, and the bradyzoites transform to the Tachyzoites, and the latent infection of Toxoplasma gondii is activated, which leads to the occurrence of fatal toxoplasmosis encephalitis (Toxoplasma gondii encephalitis). Different genotypes of Toxoplasma gondii are associated with the occurrence, development and outcome of the disease. In North America and Europe, the main genotype of Toxoplasma gondii is Type I, type II and Type III, while the dominant genotype in China is Chinese1 type. Microglia are the main innate immune components in the central nervous system, including resting and activating states, which play an important role in the pathological injury of the brain. Under the stimulation of infection and injury, microglia can activate, differentiate, proliferate and secrete a large number of neurotoxic factors, which play an immune protective role. However, excessive activation of microglia can also cause damage to peripheral tissues such as neurons. The effect of Toxoplasma gondii infection on microglia remains unclear. The aim of this study was to investigate the changes of microglial cell line (BV-2) in the infection of Toxoplasma gondii TgCtwh6 strain (Chinese1 genotype, cyst-forming strain), and to add minocycline (minocycline), a microglial inhibitor, to Toxoplasma gondii. In order to understand the inhibitory effect of Toxoplasma gondii on microglial cells and provide experimental basis for the prevention and treatment of Toxoplasma gondii encephalopathy. Methods BV-2 cells were infected with Toxoplasma gondii TgCtwh6 strain in transwell co-culture system and treated with minocycline. They were divided into three groups: the control group, the metabolite group of Toxoplasma gondii TgCtwh6 strain infection and the minocycline inhibitor group. Cell morphology was observed under inverted microscope, apoptosis was detected by flow cytometry, content of IL-1 尾 and IL-6,TNF- 偽 in cell supernatant was detected by ELISA, NO content in supernatant was measured by Griess method. The expression of IL-1 尾 and IL-6,TNF- 偽, iNOS mRNA in BV-2 cells was detected by RT-PCR assay.; Western blotting was used to detect the expression of iNOS protein in BV-2 cells. Results compared with the control group, the microglia bodies in the Toxoplasma gondii metabolite-treated group became round and the protuberances decreased. The results of flow cytometry showed that BV-2 cells infected Toxoplasma gondii for 24 hours and the ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 1. The ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 5. The apoptosis rate of BV-2 cells was 35.72%. Toxoplasma gondii metabolite-treated BV-2 cells secrete inflammatory factors, in which the contents of IL-1 尾, IL-6,TNF- 偽 and NO in culture supernatant, IL-1 尾, IL-6,TNF- 偽 in BV-2 cells, and IL-1 尾, IL-6,TNF- 偽 in BV-2 cells. The expression of iNOS mRNA and iNOS protein increased gradually with the prolongation of culture time and the ratio of Toxoplasma gondii Tachyzoites to BV-2 cells. Minocycline inhibited the production of these inflammatory factors. Conclusion Toxoplasma gondii (Toxoplasma gondii) Chinese1 genotype TgCtwh6 cystocyst metabolite can secrete inflammatory factors such as IL-1 尾 and IL-6,TNF- 偽, which may play a certain role in the process of anti-Toxoplasma gondii, which may play an important role in anti-Toxoplasma gondii. However, it also has some damage to neuron cells. Minocycline can inhibit the production of inflammatory factors, suggesting that the drug has potential therapeutic value for Toxoplasma gondii encephalopathy. However, the mechanism of Toxoplasma gondii TgCtwh6-induced inflammatory factor secretion and minocycline inhibition in microglia remains to be further studied.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R531.8
本文編號：2446499
[Abstract]:Background and objective Toxoplasma gondii is a specific intracellular parasite that infects almost all thermostat animals, including humans. Both congenital and acquired infections pose a serious threat to human health. The main manifestations of congenital toxoplasmosis were hydrocephalus, calcification, postnatal mental retardation, epilepsy and so on. Acquired toxoplasmosis can be characterized by headache fever lymph node enlargement and severe toxoplasmosis in patients with low immunity such as toxoplasmosis encephalitis. In the case of immune deficiency or hypofunction, the cyst of the brain breaks down, and the bradyzoites transform to the Tachyzoites, and the latent infection of Toxoplasma gondii is activated, which leads to the occurrence of fatal toxoplasmosis encephalitis (Toxoplasma gondii encephalitis). Different genotypes of Toxoplasma gondii are associated with the occurrence, development and outcome of the disease. In North America and Europe, the main genotype of Toxoplasma gondii is Type I, type II and Type III, while the dominant genotype in China is Chinese1 type. Microglia are the main innate immune components in the central nervous system, including resting and activating states, which play an important role in the pathological injury of the brain. Under the stimulation of infection and injury, microglia can activate, differentiate, proliferate and secrete a large number of neurotoxic factors, which play an immune protective role. However, excessive activation of microglia can also cause damage to peripheral tissues such as neurons. The effect of Toxoplasma gondii infection on microglia remains unclear. The aim of this study was to investigate the changes of microglial cell line (BV-2) in the infection of Toxoplasma gondii TgCtwh6 strain (Chinese1 genotype, cyst-forming strain), and to add minocycline (minocycline), a microglial inhibitor, to Toxoplasma gondii. In order to understand the inhibitory effect of Toxoplasma gondii on microglial cells and provide experimental basis for the prevention and treatment of Toxoplasma gondii encephalopathy. Methods BV-2 cells were infected with Toxoplasma gondii TgCtwh6 strain in transwell co-culture system and treated with minocycline. They were divided into three groups: the control group, the metabolite group of Toxoplasma gondii TgCtwh6 strain infection and the minocycline inhibitor group. Cell morphology was observed under inverted microscope, apoptosis was detected by flow cytometry, content of IL-1 尾 and IL-6,TNF- 偽 in cell supernatant was detected by ELISA, NO content in supernatant was measured by Griess method. The expression of IL-1 尾 and IL-6,TNF- 偽, iNOS mRNA in BV-2 cells was detected by RT-PCR assay.; Western blotting was used to detect the expression of iNOS protein in BV-2 cells. Results compared with the control group, the microglia bodies in the Toxoplasma gondii metabolite-treated group became round and the protuberances decreased. The results of flow cytometry showed that BV-2 cells infected Toxoplasma gondii for 24 hours and the ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 1. The ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 5. The apoptosis rate of BV-2 cells was 35.72%. Toxoplasma gondii metabolite-treated BV-2 cells secrete inflammatory factors, in which the contents of IL-1 尾, IL-6,TNF- 偽 and NO in culture supernatant, IL-1 尾, IL-6,TNF- 偽 in BV-2 cells, and IL-1 尾, IL-6,TNF- 偽 in BV-2 cells. The expression of iNOS mRNA and iNOS protein increased gradually with the prolongation of culture time and the ratio of Toxoplasma gondii Tachyzoites to BV-2 cells. Minocycline inhibited the production of these inflammatory factors. Conclusion Toxoplasma gondii (Toxoplasma gondii) Chinese1 genotype TgCtwh6 cystocyst metabolite can secrete inflammatory factors such as IL-1 尾 and IL-6,TNF- 偽, which may play a certain role in the process of anti-Toxoplasma gondii, which may play an important role in anti-Toxoplasma gondii. However, it also has some damage to neuron cells. Minocycline can inhibit the production of inflammatory factors, suggesting that the drug has potential therapeutic value for Toxoplasma gondii encephalopathy. However, the mechanism of Toxoplasma gondii TgCtwh6-induced inflammatory factor secretion and minocycline inhibition in microglia remains to be further studied.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R531.8
【共引文獻】
相關(guān)博士學(xué)位論文 前1條
1 周永華;弓形蟲慢性感染大鼠模型的建立及其在學(xué)習(xí)記憶能力影響研究中的應(yīng)用[D];南京農(nóng)業(yè)大學(xué);2010年
相關(guān)碩士學(xué)位論文 前1條
1 李冬娜;剛地弓形蟲RH株速殖子在鼠星形膠質(zhì)細胞體外培養(yǎng)的實驗觀察[D];蘇州大學(xué);2010年
本文編號：2446499
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