我国优势基因型弓形虫虫株对胎盘滋养层细胞的损伤机制研究

发布时间：2018-01-22 00:56

本文关键词： 弓形虫感染发育毒性滋养层细胞氧化应激内质网应激　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景:刚地弓形虫(Toxoplasma gondii)是专性有核细胞内寄生原虫,具有机会致病性,拥有例如众多哺乳动物,鸟类以及人类等十分广泛的宿主。弓形虫病呈世界性分布,是一种严重危害人类健康的人兽共患寄生虫病。先天性弓形虫病是人类先天畸形、神经缺陷、智力低下、死胎、早产的重要原因之一,普遍认为胎盘的损伤是引起弓形虫病的重要因素。目的:通过检测弓形虫体内感染导致胎盘氧化还原失衡与凋亡水平的变化,探讨先天性弓形虫病相关机制,为胎盘病理损伤和治疗相关疾病提供依据。方法:以受孕SPF级ICR小鼠为研究对象,随机分2组:1、正常对照组,2、WH-3株弓形虫感染组。于孕8天,感染组每只小鼠腹腔注射弓形虫毒株(WH-3株)速殖子200个/0.1mL生理盐水,对照组腹腔注射0.1mL生理盐水。孕12、14、16天分批随机颈椎脱臼处死孕鼠。孕鼠处死后,小心剥离取出胎盘,按照实验程序,制作细胞悬液用于流式细胞检测。另外,取部分胎盘液氮冻存。按照检测试剂盒操作说明,分别检测丙二醛(MDA),谷胱甘肽(GSH),8-羟基脱氧鸟苷(8-OHdG)水平的变化。流式细胞仪检测凋亡水平和活性氧簇ROS水平的变化。结果:(1)感染小鼠在孕14天后开始出现感染症状,多数在16天后开始死亡。(2)检测结果显示,实验组和正常对照组相比,孕鼠胎盘匀浆液MDA和8-OHdG含量随妊娠天数的增加而升高,GSH含量随妊娠天数的增加而降低。妊娠第12天和第14天时,实验组与正常对照组结果比较,MDA和8-OHdG含量分别从对照组的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含量从正常对照组的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)流式细胞检测分析,弓形虫感染导致胎盘滋养细胞的凋亡水平随妊娠天数的增加明显增加。孕12天,总凋亡率,早期凋亡率和晚期凋亡率分别从正常对照组的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,总凋亡率,早期凋亡率和晚期凋亡率分别从正常对照组的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%。流式检测ROS含量,孕12天感染组对正常对照组平均荧光强度分别为1034±132 vs1091±109(p0.01);孕14天感染组对正常对照组平均荧光强度分别为1036±121 vs2071±67(p0.01),均有显著性差异。(4)在0.5mM/L apocyninNADPH氧化酶特异的抑制剂处理组ROS可观察到明显减少。通过比色或免疫印迹的方法检测caspase-9,caspase-8和caspase-3的活性。研究发现,弓形虫感染组caspase-9和caspase-3的活性比caspase-8要高。结论:孕期WH-3弓形虫速殖子感染导致胎盘局部氧化应激损伤,最终引起滋养细胞的凋亡水平明显升高。氧化损伤可能是中国弓形虫分离株先天性致畸的机制之一。
[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 卤0.42 ug/gprot(12 / day and 2.57 卤0.44 ug/gprot(14 / day, respectively. 0.91 卤0.26ugP / gprott for 12 days. (0. 98 卤0. 26ugP. Gprott for 14 days) increased to 3. 13 卤0. 57 uM/gprot(12 days). 3.92 卤0.86 uM/gprot(14. 1.12 卤0.17ug-gprott for 12 days. The content of GSH in 2.65 卤0.45ugP / gprott for 14 days was 9.87 卤1.35 uM/gprot(12 in the control group. (9.88 卤1.37 UM / gprott for 14 days) decreased to 8.7 卤1.33 UM / gprot1 for 12 days). (6. 3 卤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 卤3.8and 13.1 卤2.9% to 42.0 卤7.16%, respectively. 24.8 卤6.7% and 17.2 卤2.30; The total apoptosis rate, early apoptosis rate and late apoptosis rate were 27.5 卤8.3% in the control group respectively. 14.6 卤3.3% and 12.8 卤6.3% increased to 61.2 卤9.8, 27.0 卤4.1% and 34.2 卤12.8.The ROS content was detected by flow cytometry. The average fluorescence intensity of the infected group was 1034 卤132 vs1091 卤109 vs1091 卤0.01g on the 12th day of gestation. The average fluorescence intensity of the infected group was 1036 卤121 vs2071 卤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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R531.8

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