本文關鍵詞：孕中期熱暴露影響胚胎宮內發(fā)育中HSP70的作用及其機制的初步研究　出處：《南昌大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 孕中期 熱應激 胚胎 熱休克蛋白70

【摘要】：目的:1.妊娠第8-13天對孕鼠進行熱暴露,探索孕中期熱暴露對子代宮內發(fā)育、HSP70表達及胎盤細胞凋亡的影響。2.構建SD大鼠HSP70過表達及干擾表達腺病毒載體,檢測其在SD孕鼠體內誘導或干擾循環(huán)及胎盤組織HSP70表達的效果。3.人為誘導或干擾孕鼠HSP70的表達,然后將其進行孕中期熱暴露,探討HSP70表達對熱暴露胚胎的影響及參與機制。4.高溫與子宮血流灌注不足都是FGR的重要發(fā)病因素,通過觀察比較孕中期熱暴露、子宮缺血及熱暴露后子宮缺血對胚胎和胎盤發(fā)育的影響,以及胎盤與循環(huán)HSP70的變化,探討孕中期熱暴露在FGR病因學中的影響機制。方法:1.將24只SD孕鼠隨機分為2組。對照組:孕期全程在(23±1℃)的環(huán)境中飼養(yǎng);熱暴露組:孕早期在(23±1℃)的環(huán)境中飼養(yǎng),妊娠第8天至第13天進行熱暴露,熱暴露結束后回歸(23±1℃)的環(huán)境中飼養(yǎng),在妊娠第1、8、14、21天測量并記錄孕鼠的體重。在妊娠第21天,兩組所有孕鼠用0.3%戊巴比妥麻醉后行剖腹取胎,并從腹主動脈采血3ml。測量存活胎鼠的數(shù)量、體重、性別、身長、尾長、有無畸形、胎盤重量及死胎數(shù),采用Western blotting檢測胎盤HSF70、Bax、Bc L-2的表達及ELISA檢測血漿HSP70水平。2.將12只SD孕鼠隨機分為3組。在妊娠第8天,對照組孕鼠尾靜脈注射空載腺病毒穿梭質粒,過表達組注射HSP70重組腺病毒表達載體,干擾表達組注射HSP70重組腺病毒干擾載體,各組病毒感染劑量均為2.5×109 PFU/kg。然后將三組孕鼠放置于溫度(35±1?C)的環(huán)境中進行熱暴露,感染病毒4天后處死孕鼠,采用Western blotting檢測胎盤組織HSP70蛋白表達和ELISA檢測孕鼠血漿HSP70水平以驗證過表達或干擾表達HSP70腺病毒載體的效果。3.將30只SD孕鼠隨機分為3組。在妊娠第8天,對照組尾靜脈注射空載腺病毒穿梭質粒,過表達組注射HSP70重組腺病毒表達載體,干擾表達組注射HSP70重組腺病毒干擾載體,各組病毒感染劑量均為2.5×109PFU/kg。然后進行熱暴露,妊娠第14天回歸(23±1?C)的環(huán)境中飼養(yǎng),妊娠第21天三組孕鼠用0.3%戊巴比妥麻醉后行剖腹取胎,并從腹主動脈采血3ml。測量存活胎鼠的數(shù)量、體重、性別、身長、尾長、有無畸形、胎盤重量及死胎數(shù),采用Western blotting檢測胎盤HSF70、Bax、Bc L-2的表達及ELISA法檢測血漿HSP70水平。分析胎鼠發(fā)育與胎盤、血漿HSP70表達的變化關系。4.將36只SD孕鼠隨機分為3組。熱暴露組(Ⅰ組):孕早期在(23±1℃)的環(huán)境中飼養(yǎng),妊娠第8天至第13天進行熱暴露,然后回歸(23±1℃)的環(huán)境中飼養(yǎng);子宮缺血組(Ⅱ組):孕期全程在(23±1℃)的環(huán)境中飼養(yǎng),妊娠第14天行雙側子宮動靜脈部分結扎;熱暴露后子宮缺血組(Ⅲ組):孕早期在(23±1℃)的環(huán)境中飼養(yǎng),妊娠第8天至妊娠第13天進行熱暴露,然后行雙側子宮動靜脈部分結扎后回歸(23±1℃)的環(huán)境中飼養(yǎng)。在妊娠第21天行剖腹取胎,并從腹主動脈采血3ml。測量存活胎鼠的數(shù)量、體重、性別、身長、尾長、有無畸形、胎盤重量及死胎數(shù),采用Western blotting檢測胎盤HSF70、Bax、Bc L-2的表達及ELISA法檢測血漿HSP70水平。分析胎鼠發(fā)育與胎盤、血漿HSP70表達的變化關系。結果:1.研究一結果:熱暴露組孕鼠妊娠第21天體重及孕期體重增加值均小于對照組,且差異均有統(tǒng)計學意義(均P0.05);熱暴露組胎鼠的體重、身長、尾長均小于對照組,且差異均有統(tǒng)計學意義(均P0.05);熱暴露組的胎鼠出生性別比高于對照組,差異有統(tǒng)計學意義(P0.05);熱暴露組的HSP70表達及Bax/Bcl-2比值均高于對照組,差異均有統(tǒng)計學意義(均P0.05);熱暴露組孕鼠血漿HSP70的水平高于對照組,且差異有統(tǒng)計學意義(P0.05)。2.腺病毒載體效果驗證結果:HSP70過表達載體組的循環(huán)HSP70水平高于對照組和干擾表達載體組,且差異均有統(tǒng)計學意義(P0.05),干擾HSP70表達組的血漿HSP70水平低于對照組,差異也有統(tǒng)計學意義(P0.05);HSP70過表達載體組的胎盤HSP70的表達高于對照組和干擾表達載體組,且差異均有統(tǒng)計學意義(P0.05),干擾表達載體組的胎盤HSP70表達低于對照組,差異也有統(tǒng)計學意義(P0.05)。3.研究二結果:HSP70過表達組的胎死率低于對照組和干擾表達組,且差異均有統(tǒng)計學意義(P0.05),干擾表達組與對照組胎死率的比較差異無統(tǒng)計學意義(P0.05);過表達組子代性別比最高,干擾表達組的性別比最低,且三組孕鼠子代出生性別比的比較差異均有統(tǒng)計學意義(均P0.05);HSP70過表達組的胎盤重量高于對照組與干擾表達組,且差異均有統(tǒng)計學意義(均P0.05);干擾表達組的胎盤重量低于對照組,差異同樣有統(tǒng)計學意義(P0.05);三組胎盤組織HSP70表達的水平為:過表達組最高,對照組次之,干擾表達組最低,且它們組間比較的差異均有統(tǒng)計學意義(均P0.05);三組胎盤組織Bax與Bcl-2的比值(Bax/Bcl-2)為干擾表達組最高,對照組次之,過表達組最低,且它們組間比較的差異均有統(tǒng)計學意義(均P0.05);過表達組孕鼠血漿HSP70水平低于對照組和干擾表達組,且比較差異均有統(tǒng)計學意義(均P0.05),但干擾表達組孕鼠血漿HSP70水平與對照組的比較差異無統(tǒng)計學意義(P0.05)。4.研究三結果:熱暴露后子宮缺血組孕鼠妊娠第21天體重及孕期體重增加值均低于其余兩組,且比較差異均有統(tǒng)計學意義(均P0.05),熱暴露組和子宮缺血組孕鼠妊娠第21天體重及孕期體重增加值的比較差異均無統(tǒng)計學意義(P0.05);熱暴露后子宮缺血組的胎死率高于其余兩組(均P0.05);熱暴露后子宮缺血組的胎盤重量低于其余兩組,且差異均有統(tǒng)計學意義(均P0.05);三組胎盤組織HSP70表達水平的組間比較差異均無統(tǒng)計學意義(均P0.05);三組胎盤組織Bax與Bcl-2的比值(Bax/Bcl-2)為熱暴露后子宮缺血組最高,子宮缺血組次之,熱暴露組最低,且它們組間比較的差異均有統(tǒng)計學意義(均P0.05)。熱暴露組和子宮缺血組孕鼠血漿HSP70水平無顯著差異(P0.05),但熱暴露后子宮缺血組血漿HSP70水平高于其余兩組,且差異均有統(tǒng)計學意義(均P0.05)。結論:1.孕中期(35±1℃)熱暴露會給孕鼠子代的宮內生長發(fā)育帶來不利影響。2.p AD-CMV-HSP70重組腺病毒表達載體與U6-sh RNA-loop-anti-sh RNAteminator-UBC-e GFP-HSP70重組腺病毒干擾載體用于提高或干擾孕鼠循環(huán)及胎盤HSP70的表達是可行的。3.誘導孕中期熱暴露孕鼠HSP70高表達可以有助于胎盤的發(fā)育,降低胎死率;干擾孕中期熱暴露孕鼠HSP70表達則會影響胎盤的生長發(fā)育。4.胎盤HSP70表達水平與子代胎死率密切相關,推測胎盤HSP70高表達具有促進胎盤生長及提高胚胎存活率的作用。5.孕中期熱暴露后子宮缺血會嚴重影響胎盤及胚胎的宮內發(fā)育。與細胞內HSP70不同,母體循環(huán)中HSP70水平的升高會給胚胎的發(fā)育帶來不利影響。
[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,韜暱,灝鵑暱,鏈夋棤鐣稿艦,鑳庣洏閲嶉噺鍙婃鑳庢暟,閲囩敤Western blotting媯，

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