本文關鍵詞： 弓形蟲感染 發(fā)育毒性 滋養(yǎng)層細胞 氧化應激 內(nèi)質網(wǎng)應激　出處：《安徽醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：背景:剛地弓形蟲(Toxoplasma gondii)是專性有核細胞內(nèi)寄生原蟲,具有機會致病性,擁有例如眾多哺乳動物,鳥類以及人類等十分廣泛的宿主。弓形蟲病呈世界性分布,是一種嚴重危害人類健康的人獸共患寄生蟲病。先天性弓形蟲病是人類先天畸形、神經(jīng)缺陷、智力低下、死胎、早產(chǎn)的重要原因之一,普遍認為胎盤的損傷是引起弓形蟲病的重要因素。目的:通過檢測弓形蟲體內(nèi)感染導致胎盤氧化還原失衡與凋亡水平的變化,探討先天性弓形蟲病相關機制,為胎盤病理損傷和治療相關疾病提供依據(jù)。方法:以受孕SPF級ICR小鼠為研究對象,隨機分2組:1、正常對照組,2、WH-3株弓形蟲感染組。于孕8天,感染組每只小鼠腹腔注射弓形蟲毒株(WH-3株)速殖子200個/0.1mL生理鹽水,對照組腹腔注射0.1mL生理鹽水。孕12、14、16天分批隨機頸椎脫臼處死孕鼠。孕鼠處死后,小心剝離取出胎盤,按照實驗程序,制作細胞懸液用于流式細胞檢測。另外,取部分胎盤液氮凍存。按照檢測試劑盒操作說明,分別檢測丙二醛(MDA),谷胱甘肽(GSH),8-羥基脫氧鳥苷(8-OHdG)水平的變化。流式細胞儀檢測凋亡水平和活性氧簇ROS水平的變化。結果:(1)感染小鼠在孕14天后開始出現(xiàn)感染癥狀,多數(shù)在16天后開始死亡。(2)檢測結果顯示,實驗組和正常對照組相比,孕鼠胎盤勻漿液MDA和8-OHdG含量隨妊娠天數(shù)的增加而升高,GSH含量隨妊娠天數(shù)的增加而降低。妊娠第12天和第14天時,實驗組與正常對照組結果比較,MDA和8-OHdG含量分別從對照組的2.52±0.42 ug/gprot(12天)、2.57±0.44 ug/gprot(14天);0.91±0.26ug/gprot(12天)、0.98±0.26ug/gprot(14天)升高到3.13±0.57 uM/gprot(12天)、3.92±0.86 uM/gprot(14天);1.12±0.17ug/gprot(12天)、2.65±0.45ug/gprot(14天)(p0.05)。GSH含量從正常對照組的9.87±1.35 uM/gprot(12天)、9.88±1.37uM/gprot(14天)降低到8.7±1.33uM/gprot(12天)、6.3±1.2 uM/gprot(14天)(p0.01)。(3)流式細胞檢測分析,弓形蟲感染導致胎盤滋養(yǎng)細胞的凋亡水平隨妊娠天數(shù)的增加明顯增加。孕12天,總凋亡率,早期凋亡率和晚期凋亡率分別從正常對照組的27.1±3.8%,14.0±2.7%和13.1±2.9%上升到42.0±7.16%,24.8±6.7%和17.2±2.30%;孕14天,總凋亡率,早期凋亡率和晚期凋亡率分別從正常對照組的27.5±8.3%,14.6±3.3%和12.8±6.3%上升到61.2±9.8%,27.0±4.1%和34.2±12.8%。流式檢測ROS含量,孕12天感染組對正常對照組平均熒光強度分別為1034±132 vs1091±109(p0.01);孕14天感染組對正常對照組平均熒光強度分別為1036±121 vs2071±67(p0.01),均有顯著性差異。(4)在0.5mM/L apocyninNADPH氧化酶特異的抑制劑處理組ROS可觀察到明顯減少。通過比色或免疫印跡的方法檢測caspase-9,caspase-8和caspase-3的活性。研究發(fā)現(xiàn),弓形蟲感染組caspase-9和caspase-3的活性比caspase-8要高。結論:孕期WH-3弓形蟲速殖子感染導致胎盤局部氧化應激損傷,最終引起滋養(yǎng)細胞的凋亡水平明顯升高。氧化損傷可能是中國弓形蟲分離株先天性致畸的機制之一。
[Abstract]:Background: Toxoplasma gondii (Toxoplasma gondii) is a parasitic protozoa with specific nucleated cells, which has opportunistic pathogenicity, such as many mammals. Toxoplasma gondii is a serious human and zoonotic parasitic disease, which is a worldwide distribution. Congenital toxoplasmosis is human congenital malformation, nerve defects. Mental retardation, stillbirth, preterm birth is one of the important reasons. It is generally believed that placental injury is an important factor in Toxoplasma gondii. Objective: to investigate the mechanism of congenital toxoplasmosis by detecting the changes of placental redox imbalance and apoptosis caused by Toxoplasma gondii infection in vivo. Methods: pregnant SPF grade ICR mice were randomly divided into 2 groups: 1, normal control group. WH-3 strain Toxoplasma gondii infection group. On the 8th day of pregnancy, each mouse in the infected group was injected intraperitoneally with Toxoplasma gondii strain WH-3) 200 / 0.1 mL normal saline of Toxoplasma gondii Toxoplasma gondii. In the control group, 0.1 mL saline was injected intraperitoneally. The pregnant mice were killed by random cervical dislocations on the 12th day of gestation. After the pregnant rats were killed, the placenta was removed carefully and the placenta was taken out according to the experimental procedure. Cell suspensions were prepared for flow cytometry. In addition, some placental liquid nitrogen was frozen. According to the operating instructions of the test kit, malondialdehyde (MDA) and glutathione (GSH) were detected respectively. Changes of 8-OHdG level of 8-hydroxydeoxyguanosine. Flow cytometry was used to detect the changes of apoptosis level and ROS level of reactive oxygen species. The infected mice began to develop infection symptoms after 14 days of pregnancy. The results showed that the levels of MDA and 8-OHdG in placenta homogenate of pregnant rats increased with the increase of gestational days. The content of GSH decreased with the increase of gestational days. On the 12th and 14th day of pregnancy, the results of the experimental group were compared with those of the normal control group. The contents of MDA and 8-OHdG were 2.52 鹵0.42 ug/gprot(12 / day and 2.57 鹵0.44 ug/gprot(14 / day, respectively. 0.91 鹵0.26ugP / gprott for 12 days. (0. 98 鹵0. 26ugP. Gprott for 14 days) increased to 3. 13 鹵0. 57 uM/gprot(12 days). 3.92 鹵0.86 uM/gprot(14. 1.12 鹵0.17ug-gprott for 12 days. The content of GSH in 2.65 鹵0.45ugP / gprott for 14 days was 9.87 鹵1.35 uM/gprot(12 in the control group. (9.88 鹵1.37 UM / gprott for 14 days) decreased to 8.7 鹵1.33 UM / gprot1 for 12 days). (6. 3 鹵1. 2 uM/gprot(14) flow cytometry analysis. Toxoplasma gondii infection caused the apoptosis of trophoblast in placenta increased with the increase of gestational days. The rate of early and late apoptosis increased from 27.1 鹵3.8and 13.1 鹵2.9% to 42.0 鹵7.16%, respectively. 24.8 鹵6.7% and 17.2 鹵2.30; The total apoptosis rate, early apoptosis rate and late apoptosis rate were 27.5 鹵8.3% in the control group respectively. 14.6 鹵3.3% and 12.8 鹵6.3% increased to 61.2 鹵9.8, 27.0 鹵4.1% and 34.2 鹵12.8.The ROS content was detected by flow cytometry. The average fluorescence intensity of the infected group was 1034 鹵132 vs1091 鹵109 vs1091 鹵0.01g on the 12th day of gestation. The average fluorescence intensity of the infected group was 1036 鹵121 vs2071 鹵67p 0.01 on the 14th day of gestation. There was significant difference between the two groups. The decrease of ROS was observed in 0.5 mm / L apocyninNADPH oxidase specific inhibitor treatment group. Caspase-9 was detected by colorimetry or Western blotting. Activity of caspase-8 and caspase-3. The activity of caspase-9 and caspase-3 in Toxoplasma gondii infection group was higher than that in caspase-8 group. The infection of Toxoplasma gondii Tachyzoites during pregnancy caused local oxidative stress injury in placenta. Oxidative damage may be one of the mechanisms of congenital teratogenicity of Toxoplasma gondii isolates in China.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R531.8

【共引文獻】

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本文編號：1453103