母体孕期镉暴露诱发胎儿神经管畸形和生长受限的机制

发布时间：2018-04-01 22:02

  本文选题：镉　切入点：神经管畸形　出处：《安徽医科大学》2017年硕士论文

【摘要】：目的:此前的研究发现孕鼠在孕期GD9暴露镉可致胎鼠前肢缺趾畸形和尾部畸形,本次实验研究在胎儿神经管形成期对孕鼠进行镉暴露是否会导致胎鼠神经管畸形并初步探讨其部分机制。方法:本次实验主要包括以下三个实验:实验一:镉致胎鼠神经管畸形模型:36只ICR小鼠在受孕第8(GD8)天按体重随机平均分为三组,于GD8天8:00a.m-9:00a.m通过腹腔注射分别给予0、2.5、5mg/kg的Cd Cl_2。受孕期间三天称量一次孕鼠体重,每天记录孕鼠饮食饮水量。GD18天进行取材,摘眼球法收集孕鼠血液,颈椎脱臼法处死并剖杀所有孕鼠,记录活胎数、死胎数、畸形胎鼠数和吸收胎数,称量活胎体重和胎盘重量,留取胎盘作病理切片观察HE染色。实验二:研究孕鼠镉暴露后对叶酸转运的影响:24只ICR小鼠按体重在受孕第8(GD8)天按体重随机分为两组(对照组与镉处理组),镉处理组于GD8天8:00a.m-9:00a.m单次腹腔注射5mg/kg的Cd Cl_2,对照组给予生理盐水处理。24h后剖杀孕鼠,摘眼球法收集孕鼠血液,取胚胎和胎盘。血液室温静置2h左右3000转离心10min留血清和胚胎一起-80℃保存用于检测叶酸含量。实验三:研究孕鼠镉暴露后对胎盘叶酸转运泵的影响:48只ICR小鼠按体重在受孕第8(GD8)天按体重随机分为8组。其中,镉处理组于GD8天8:00a.m-9:00a.m单次腹腔注射5mg/kg的Cd Cl_2,对照组给予生理盐水处理。镉处理组孕鼠于镉处理后不同时间点剖杀(2h、12h、24h、48h、72h),对照组于0h、24h、48h、72h进行剖杀。摘眼球法收集孕鼠血液,取胎盘做RT-PCR和Western Blotting实验检测胎盘叶酸转运泵水平。结果:孕鼠孕期第8天暴露镉可致胎鼠神经管畸形。其中2.5mg/kg镉处理组的窝神经管畸形率为33.3%(4/12),平均胎鼠神经管畸形率为3.6%,5mg/kg镉处理组的窝神经管畸形率高达75%(9/12),平均胎鼠神经管畸形率为20.0%,同时镉暴露可致胎鼠体重减少,母体孕期镉暴露对胎鼠体重的减少呈剂量反应关系(剂量越高,胎鼠体重越轻)。此外,镉暴露也会导致胎鼠身长明显减少;并且孕鼠孕期镉暴露会损害胎盘的发育,对胎盘重量的减少呈剂量反应关系(剂量越高,胎盘重量越轻)。相应的镉暴露也会导致胎盘直径明显减少,对胎盘的病理HE染色发现,镉处理组的胎盘迷路层血管明显减少。我们进一步检测了母体血清和胎鼠的叶酸水平,发现镉处理组的胎盘叶酸水平明显下降;此后,我们检测了胎盘的叶酸转运泵Pcft,Frα和Rfc1,发现Pcft的m RNA水平在镉处理2h后就显著下调,并且在镉处理24h后依旧保持下降状态,我们通过Weatern Blotting实验在蛋白水平也得到了验证。结论:孕鼠妊娠期在胎儿器官形成阶段进行镉暴露,会下调胎盘的叶酸转运泵的表达,从而干扰叶酸由母体循环进入胎儿体内继而诱发胎鼠神经管畸形。
[Abstract]:Objective: previous studies have found that exposure to cadmium in GD9 during pregnancy can lead to deformity of forelimb and tail of fetal mice. This experiment was conducted to study whether exposure to cadmium in fetal rats during fetal neural tube formation would lead to fetal neural tube malformation and explore some of its mechanisms. Methods: this experiment mainly includes the following three experiments: experiment 1: cadmium induced by cadmium. Fetal rat neural tube malformation model: 36 ICR mice were randomly divided into three groups according to their body weight on the 8th day of conception. On the day of GD8, 8:00a.m-9:00a.m was injected intraperitoneally with 0.5 mg / kg of CD Cl2.The body weight of pregnant rats was measured once every three days during pregnancy, the diet and drinking quantity of pregnant rats were recorded every day for 18 days, the blood of pregnant rats was collected by eyeball extraction, the cervical vertebrae dislocated and all pregnant rats were killed and all the pregnant rats were killed. The number of live fetus, the number of dead fetus, the number of malformed fetus and the number of absorbed fetus, the weight of live fetus and placenta weight were recorded. The effect of cadmium exposure on folic acid transport in pregnant mice was studied. Experiment 2: 24 ICR mice were randomly divided into two groups according to their body weight according to their body weight on the 8th day of gestation (control group and cadmium treatment group). The rats in the control group were treated with normal saline for 24 hours. The pregnant rats were killed by single intraperitoneal injection of CDCl2 from 8:00a.m-9:00a.m on the day of GD8, and the rats in the control group were treated with normal saline for 24 hours. The blood of pregnant mice was collected by eyeball extraction. Fetuses and placentas were taken. Blood was stored at room temperature for 2 hours or so for 3000 rpm centrifugation 10min to preserve serum and embryo together at -80 鈩，

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