孕中期热暴露影响胚胎宫内发育中HSP70的作用及其机制的初步研究

发布时间：2018-01-17 21:28

  本文关键词：孕中期热暴露影响胚胎宫内发育中HSP70的作用及其机制的初步研究　出处：《南昌大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 孕中期 热应激 胚胎 热休克蛋白70

【摘要】：目的:1.妊娠第8-13天对孕鼠进行热暴露,探索孕中期热暴露对子代宫内发育、HSP70表达及胎盘细胞凋亡的影响。2.构建SD大鼠HSP70过表达及干扰表达腺病毒载体,检测其在SD孕鼠体内诱导或干扰循环及胎盘组织HSP70表达的效果。3.人为诱导或干扰孕鼠HSP70的表达,然后将其进行孕中期热暴露,探讨HSP70表达对热暴露胚胎的影响及参与机制。4.高温与子宫血流灌注不足都是FGR的重要发病因素,通过观察比较孕中期热暴露、子宫缺血及热暴露后子宫缺血对胚胎和胎盘发育的影响,以及胎盘与循环HSP70的变化,探讨孕中期热暴露在FGR病因学中的影响机制。方法:1.将24只SD孕鼠随机分为2组。对照组:孕期全程在(23±1℃)的环境中饲养;热暴露组:孕早期在(23±1℃)的环境中饲养,妊娠第8天至第13天进行热暴露,热暴露结束后回归(23±1℃)的环境中饲养,在妊娠第1、8、14、21天测量并记录孕鼠的体重。在妊娠第21天,两组所有孕鼠用0.3%戊巴比妥麻醉后行剖腹取胎,并从腹主动脉采血3ml。测量存活胎鼠的数量、体重、性别、身长、尾长、有无畸形、胎盘重量及死胎数,采用Western blotting检测胎盘HSF70、Bax、Bc L-2的表达及ELISA检测血浆HSP70水平。2.将12只SD孕鼠随机分为3组。在妊娠第8天,对照组孕鼠尾静脉注射空载腺病毒穿梭质粒,过表达组注射HSP70重组腺病毒表达载体,干扰表达组注射HSP70重组腺病毒干扰载体,各组病毒感染剂量均为2.5×109 PFU/kg。然后将三组孕鼠放置于温度(35±1?C)的环境中进行热暴露,感染病毒4天后处死孕鼠,采用Western blotting检测胎盘组织HSP70蛋白表达和ELISA检测孕鼠血浆HSP70水平以验证过表达或干扰表达HSP70腺病毒载体的效果。3.将30只SD孕鼠随机分为3组。在妊娠第8天,对照组尾静脉注射空载腺病毒穿梭质粒,过表达组注射HSP70重组腺病毒表达载体,干扰表达组注射HSP70重组腺病毒干扰载体,各组病毒感染剂量均为2.5×109PFU/kg。然后进行热暴露,妊娠第14天回归(23±1?C)的环境中饲养,妊娠第21天三组孕鼠用0.3%戊巴比妥麻醉后行剖腹取胎,并从腹主动脉采血3ml。测量存活胎鼠的数量、体重、性别、身长、尾长、有无畸形、胎盘重量及死胎数,采用Western blotting检测胎盘HSF70、Bax、Bc L-2的表达及ELISA法检测血浆HSP70水平。分析胎鼠发育与胎盘、血浆HSP70表达的变化关系。4.将36只SD孕鼠随机分为3组。热暴露组(Ⅰ组):孕早期在(23±1℃)的环境中饲养,妊娠第8天至第13天进行热暴露,然后回归(23±1℃)的环境中饲养;子宫缺血组(Ⅱ组):孕期全程在(23±1℃)的环境中饲养,妊娠第14天行双侧子宫动静脉部分结扎;热暴露后子宫缺血组(Ⅲ组):孕早期在(23±1℃)的环境中饲养,妊娠第8天至妊娠第13天进行热暴露,然后行双侧子宫动静脉部分结扎后回归(23±1℃)的环境中饲养。在妊娠第21天行剖腹取胎,并从腹主动脉采血3ml。测量存活胎鼠的数量、体重、性别、身长、尾长、有无畸形、胎盘重量及死胎数,采用Western blotting检测胎盘HSF70、Bax、Bc L-2的表达及ELISA法检测血浆HSP70水平。分析胎鼠发育与胎盘、血浆HSP70表达的变化关系。结果:1.研究一结果:热暴露组孕鼠妊娠第21天体重及孕期体重增加值均小于对照组,且差异均有统计学意义(均P0.05);热暴露组胎鼠的体重、身长、尾长均小于对照组,且差异均有统计学意义(均P0.05);热暴露组的胎鼠出生性别比高于对照组,差异有统计学意义(P0.05);热暴露组的HSP70表达及Bax/Bcl-2比值均高于对照组,差异均有统计学意义(均P0.05);热暴露组孕鼠血浆HSP70的水平高于对照组,且差异有统计学意义(P0.05)。2.腺病毒载体效果验证结果:HSP70过表达载体组的循环HSP70水平高于对照组和干扰表达载体组,且差异均有统计学意义(P0.05),干扰HSP70表达组的血浆HSP70水平低于对照组,差异也有统计学意义(P0.05);HSP70过表达载体组的胎盘HSP70的表达高于对照组和干扰表达载体组,且差异均有统计学意义(P0.05),干扰表达载体组的胎盘HSP70表达低于对照组,差异也有统计学意义(P0.05)。3.研究二结果:HSP70过表达组的胎死率低于对照组和干扰表达组,且差异均有统计学意义(P0.05),干扰表达组与对照组胎死率的比较差异无统计学意义(P0.05);过表达组子代性别比最高,干扰表达组的性别比最低,且三组孕鼠子代出生性别比的比较差异均有统计学意义(均P0.05);HSP70过表达组的胎盘重量高于对照组与干扰表达组,且差异均有统计学意义(均P0.05);干扰表达组的胎盘重量低于对照组,差异同样有统计学意义(P0.05);三组胎盘组织HSP70表达的水平为:过表达组最高,对照组次之,干扰表达组最低,且它们组间比较的差异均有统计学意义(均P0.05);三组胎盘组织Bax与Bcl-2的比值(Bax/Bcl-2)为干扰表达组最高,对照组次之,过表达组最低,且它们组间比较的差异均有统计学意义(均P0.05);过表达组孕鼠血浆HSP70水平低于对照组和干扰表达组,且比较差异均有统计学意义(均P0.05),但干扰表达组孕鼠血浆HSP70水平与对照组的比较差异无统计学意义(P0.05)。4.研究三结果:热暴露后子宫缺血组孕鼠妊娠第21天体重及孕期体重增加值均低于其余两组,且比较差异均有统计学意义(均P0.05),热暴露组和子宫缺血组孕鼠妊娠第21天体重及孕期体重增加值的比较差异均无统计学意义(P0.05);热暴露后子宫缺血组的胎死率高于其余两组(均P0.05);热暴露后子宫缺血组的胎盘重量低于其余两组,且差异均有统计学意义(均P0.05);三组胎盘组织HSP70表达水平的组间比较差异均无统计学意义(均P0.05);三组胎盘组织Bax与Bcl-2的比值(Bax/Bcl-2)为热暴露后子宫缺血组最高,子宫缺血组次之,热暴露组最低,且它们组间比较的差异均有统计学意义(均P0.05)。热暴露组和子宫缺血组孕鼠血浆HSP70水平无显著差异(P0.05),但热暴露后子宫缺血组血浆HSP70水平高于其余两组,且差异均有统计学意义(均P0.05)。结论:1.孕中期(35±1℃)热暴露会给孕鼠子代的宫内生长发育带来不利影响。2.p AD-CMV-HSP70重组腺病毒表达载体与U6-sh RNA-loop-anti-sh RNAteminator-UBC-e GFP-HSP70重组腺病毒干扰载体用于提高或干扰孕鼠循环及胎盘HSP70的表达是可行的。3.诱导孕中期热暴露孕鼠HSP70高表达可以有助于胎盘的发育,降低胎死率;干扰孕中期热暴露孕鼠HSP70表达则会影响胎盘的生长发育。4.胎盘HSP70表达水平与子代胎死率密切相关,推测胎盘HSP70高表达具有促进胎盘生长及提高胚胎存活率的作用。5.孕中期热暴露后子宫缺血会严重影响胎盘及胚胎的宫内发育。与细胞内HSP70不同,母体循环中HSP70水平的升高会给胚胎的发育带来不利影响。
[Abstract]:Objective: 1. days 8-13 of pregnancy in pregnant rats by heat exposure, explore the second trimester heat exposure on offspring intrauterine growth, effect of.2. HSP70 expression and apoptosis of placental cells to construct SD rat HSP70 overexpression and RNAi adenovirus vector, detecting the expression of human induced or interference in pregnant HSP70 rats.3. effect in pregnant mice SD the induction or interference of HSP70 circulation and placental tissue expression, and then the second trimester heat exposure, the expression of HSP70 and FGR are important factors in the pathogenesis of heat exposure and the effect of embryonic participation mechanism of.4. high temperature and uterine hypoperfusion, through observation and comparison of second trimester exposure to heat, impact on the development of embryo and placenta uterine ischemia the uterine ischemia and after thermal exposure, and the changes of placenta and circulating HSP70, to investigate the effects and mechanism of FGR in the etiology of second trimester exposure. Methods: 1. of 24 pregnant SD rats were randomly divided into 2 groups: pregnant control group. In the whole period (23 - 1 DEG C) feeding environment; heat exposure group: in early pregnancy (23 + - 1 DEG C) feeding environment, pregnancy eighth days to thirteenth days of heat exposure, exposure to heat after regression (23 + - 1 DEG C) feeding environment, in 1,8,14,21 days of pregnancy and measurement record the weight of pregnant rats. In the twenty-first day of pregnancy. All two groups of pregnant rats with 0.3% pentobarbital anesthesia after caesarean section, and from the abdominal aorta 3ml. measuring the number of survival, fetal weight, sex, body length, tail length, there is no deformity, placental weight and number of stillbirth, placental blotting detection using Western HSF70, Bax,.2. expression and ELISA detection of the plasma level of HSP70 L-2 will Bc 12 pregnant SD rats were randomly divided into 3 groups. In the eighth day of pregnancy, pregnant rats control group intravenous injection of empty adenovirus shuttle plasmid expression group were injected with HSP70 recombinant adenovirus expression vector, expression of interference group injection of recombinant adenovirus HSP70 virus load disturbance The body, each virus infection dose was 2.5 * 109 PFU/kg. and then three groups of pregnant rats were placed in temperature (35 + 1? C) were exposed to heat environment, pregnant rats were sacrificed 4 days after virus infection, using Western blotting detected the expression of HSP70 protein and ELISA in placenta of pregnant rats were measured to verify the expression level of plasma HSP70 the expression of.3. or interference effect of HSP70 adenovirus vector of 30 pregnant SD rats were randomly divided into 3 groups. In the eighth day of pregnancy, the control group were injected with empty adenovirus shuttle plasmid expression group were injected with HSP70 recombinant adenovirus expression vector expressing adenovirus interference interference vector group were injected with recombinant HSP70 virus infection dose groups both 2.5 * 109PFU/kg. and heat exposure, the fourteenth day of pregnancy regression (23 + 1? C) feeding environment, twenty-first days of three groups of pregnant mice with 0.3% pentobarbital anesthesia after caesarean section, and the blood from the abdominal aorta to measure fetal survival 3ml. 榧犵殑鏁伴噺,浣撻噸,鎬у埆,韬�闀�,灏鹃暱,鏈夋棤鐣稿舰,鑳庣洏閲嶉噺鍙婃�昏儙鏁�,閲囩敤Western blotting妫，

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