妊娠期正己烷暴露后子代卵巢发育及其细胞DNA甲基化模式研究

发布时间：2018-04-23 05:39

本文选题：正己烷 + 妊娠期暴露　；参考：《福建医科大学》2013年博士论文

【摘要】：正己烷是一种广泛用于食品、药品和有机合成的溶剂。因其常温下易挥发职业暴露多通过呼吸道途径，暴露人群中育龄女性比例较高。正己烷早期被认为低毒或微毒物而广泛应用于各行业，但近年来的研究表明正己烷及其代谢产物对雌性生殖内分泌有明显的干扰，同时基础毒理学资料表明妊娠期正己烷暴露有可能造成子代生长发育障碍，且正己烷无明显的致DNA突变效应。因此我们通过建立妊娠Wista大鼠正己烷暴露模型来研究妊娠期正己烷暴露对子代成年后卵巢生长发育、卵巢颗粒细胞DNA甲基化状态及卵巢颗粒细胞激素分泌功能的影响，明确胚胎发育期正己烷暴露对子代雌性生殖系统发育的影响，有利于进一步提高对正己烷职业暴露的防控和危险人群的干预。目的：通过建立Wista大鼠妊娠期正己烷暴露模型，研究妊娠期正己烷暴露对子代成年后卵巢生长发育、卵巢颗粒细胞DNA甲基化状态及卵巢颗粒细胞激素分泌功能的影响，进一步从机制上阐明妊娠期正己烷暴露对于子代卵巢发育和功能的影响。为正己烷卵巢发育毒性及机制研究提供科学依据。基于以上目的，我们将研究分为以下三个部分：（一）妊娠期正己烷暴露对F1代大鼠卵巢发育和激素分泌功能的影响方法：清洁级成年Wistar大鼠，雌性体重210-230g，雄性300-320g，合笼受孕后，每组5只。GD1-20分别暴露于0、100、500、2500和12500ppm正己烷，静式吸入染毒，4h/d，F1代雌鼠PD56取卵巢。观察大鼠的出生性别比、F1代雌鼠的体重增长、阴道开放时间和动情周期；HE染色连续切片测量各级卵泡数并观察卵巢病理改变；分离F1代雌鼠卵巢颗粒细胞，竞争性电化学发光法测定F1代雌鼠卵巢颗粒细胞培养液雌二醇和孕酮水平。结果：（1）出生活胎数和性别比：12500ppm组的出生活胎数显著低于其他各组（p0.05）；随着暴露剂量的增加，雌/雄性别比有下降的趋势。（2）体重增长和阴道开放时间：各正己烷暴露组体重和对照组总体上无显著性差异（p0.05），但d36日之后有增长减缓的趋势；正己烷暴露组阴道开放时间与对照组比较无显著差异（p0.05）。（3）动情周期：与对照组比较，100，500和2500ppm组的动情前期显著延长（p0.05），500ppm和2500ppm组动情期显著延长（p0.05），12500ppm组动情间期显著缩短（p0.05）。（4）卵泡数目构成比：12500ppm组的次级卵泡比例显著低于对照组（p0.05），闭锁卵泡比例显著高于对照组（p0.05）。（5）卵巢颗粒细胞培养液雌二醇、孕酮水平：2500和12500ppm雌二醇分泌水平显著低于对照组（p0.05）；100和500ppm组孕酮分泌水平显著高于对照组，而12500ppm组孕酮分泌水平显著低于对照组（p0.05）。（二）妊娠期正己烷暴露对F1代大鼠卵巢颗粒细胞启动子区差异甲基化全基因组研究方法：分离F1代大鼠的卵巢颗粒细胞，DNeasyBloodTissueKit提取基因组DNA，超声破碎后与5-mC抗体免疫共沉淀，再与Nimblegen385KPromoterPlusCpGIslandArrays杂交，以启动子区探针PeakScore2并和对照组有差别为标准筛选差异甲基化基因。将差异甲基化基因导入DAVID数据库进行以下分析：GeneOntologyConsortium方法注释差异性甲基化基因、差异性甲基化基因DAVID聚类分析、DAVID聚类基因Pmvalue值二项聚类分析组间甲基化模式差异、差异甲基化基因KEGG信号通路分析。结果：（1）基因组DNA无明显的降解并裂解充分，甲基化免疫沉淀特异性高，，甲基化芯片信号清楚，阳性对照信号清晰。（2）正己烷暴露组共同基因中，去甲基化基因多于高甲基化基因。（3）基因注释表明正己烷的效应主要与细胞过程、代谢过程、细胞内基因和磷酸转移酶活性基因有关。（4）细胞死亡、细胞生长和激素调控三个DAVID聚类结果pm值分析结果表明，对照和500ppm组的甲基化模式比较接近，2500和12500ppm组的甲基化模式相对接近。（5）KEGG信号通路分析结果的表明：凋亡通路中12500ppm组促凋亡基因Bad启动子区低甲基化，500ppm组凋亡促进基因NFKBIA，Bid，Casp7，Dffb启动子区高甲基化。在激素合成通路，2500和12500ppm组Cyp11a1,Cyp17a1,Hsd3b1和Srd5a1基因启动子区的高甲基化。（三）妊娠期正己烷暴露对F1代大鼠卵巢颗粒细胞激素分泌相关基因启动子甲基化研究方法：测定MeDIP-Chip芯片谱中激素合成基因相关基因启动子区差异甲基化峰及探针位点，MS-HRM法验证筛选出激素合成相关基因启动子区甲基化状态，RealtimePCR法测定筛选基因的mRNA表达水平。结果：（1）激素合成基因相关基因启动子区差异甲基化峰测量：Star基因，500ppm组的甲基化程度低于对照组，而12500ppm组明显高于对照组（p0.05）；Cyp11a1基因，2500ppm和12500ppm组的甲基化程度明显高于对照组（p0.05）；Cyp17a1基因，2500ppm和12500ppm组的甲基化程度明显高于对照组（p0.05）；Hsd3b基因，12500ppm组的甲基化程度显著高于对照组（p0.05）。（2）MS-HRM验证甲基化程度：Star基因启动子局部区域甲基化程度从高到低排列为：12500ppm2500ppmcontrol500ppm100ppm；Cyp11a1基因启动子局部区域甲基化程度从高到低排列为：12500ppm2500ppmcontrol500ppm和100ppm；Cyp17a1启动子局部区域甲基化程度从高到低排列为：12500ppm和2500ppmcontrol500ppm和100ppm；Hsd3b启动子局部区域基因甲基化程度从高到低排列为：12500ppm2500ppm和Control100ppm和500ppm。（3）RT-PCR结果：Star、Cyp11a1、Cyp17a1和Hsd3bmRNA表达量与对照组比较出现了明显改变，具体如下：与对照组比较，Star基因，500ppm组表达量显著增高（p0.05），12500ppm组表达量显著下降（p0.05）；Cyp11a1基因，500ppm组表达量显著增高（p0.05），12500ppm表达量显著下降（p0.05）；Cyp17a1基因，500ppm组显著增高（p0.05），2500ppm和12500ppm显著下降（p0.05）；Hsd3b基因，12500ppm组显著下降（p0.05）。结论：根据以上结果，可得出如下结论 1.妊娠期高浓度正己烷暴露具有明显的胚胎毒性，并能影响F1代雌性大鼠的卵巢发育和激素分泌功能。 2.妊娠期正己烷暴露会改变子代卵巢颗粒细胞的DNA启动子区甲基化状态，高剂量的正己烷暴露（2500和12500ppm）能明显改变颗粒细胞凋亡基因和激素合成基因的甲基化状态。 3.妊娠期正己烷暴露后，卵巢颗粒细胞中激素合成相关基因Star、Cyp11a1、Cyp17a1和Hsd3b的启动子局部区域的甲基化状态改变与对应基因mRNA水平表达异常，性激素分泌能力改变可能存在一定的联系。
[Abstract]:n - hexane ( n - hexane ) is a widely used solvent for food , medicine and organic synthesis .

Purpose :

The effects of n - hexane exposure during pregnancy on the growth and development of ovary , the DNA methylation status of ovary granular cells and the function of ovarian granular cell hormone secretion were studied by establishing a normal hexane exposure model in pregnancy . The effects of n - hexane exposure during pregnancy on the development and function of ovary were further elucidated .

The effects of n - hexane exposure during pregnancy on ovarian development and hormone secretion in F1 generation rats

Method :

Male Wistar rats weighing 210 - 230g and male 300 - 320g were exposed to 0 , 100 , 500 , 2500 , and 12500ppm n - hexane respectively .
HE stained continuous sections to measure the number of follicles at all levels and observe the pathological changes of the ovary ;
The levels of estradiol and progesterone in the ovary granular cells of F1 generation female mice were determined by competitive electrochemical luminescence .

Results :

( 1 ) The number of live fetuses and sex ratio were significantly lower than those in other groups ( p < 0.05 ) .
With the increase of exposure dose , the ratio of female / male sex ratio decreased .

( 2 ) Body weight gain and vaginal opening time : There was no significant difference between the body weight of each n - hexane exposure group and the control group ( p < 0.05 ) , but there was a tendency of slowing down after d36 days .
The vaginal opening time of n - hexane exposed group was not significantly different from that of the control group ( p < 0.05 ) .

( 3 ) The estrus cycle : Compared with the control group , the estrus period of the 100 , 500 and 2500 ppm groups was significantly prolonged ( p < 0.05 ) , the dynamic period of 500 ppm and 2500 ppm group was significantly prolonged ( p < 0.05 ) , and the estrus interval in the 12500 ppm group was significantly shortened ( p < 0.05 ) .

( 4 ) The proportion of follicles in the follicles was significantly lower than that in the control group ( p < 0.05 ) , and the proportion of follicles in the follicles was significantly higher than that of the control group ( p < 0.05 ) .

( 5 ) The levels of estradiol and progesterone in ovarian granular cell culture fluid were significantly lower than those in control group ( p < 0.05 ) .
The levels of progesterone secretion in the 100 and 500 ppm groups were significantly higher than those in the control group , while the level of progesterone secretion in the 12500 ppm group was significantly lower than that of the control group ( p < 0.05 ) .

( 2 ) Whole - gene group of differential methylation of normal hexane exposure during pregnancy on the promoter region of ovary granular cell in F1 generation rat

Method :

The genomic DNA was isolated from F1 generation rats . The genomic DNA was extracted by DNeaster TissueKit , and then hybridized with 5 - mC antibody after ultrasonic disruption . The differential methylated gene was introduced into DAVID database . The difference methylation gene was introduced into DAVID database . The difference of methylation patterns between the two cluster analysis groups was analyzed by GeneOntologyConsortium method .

Results :

( 1 ) the genome DNA has no obvious degradation and lysis , the methylation immunoprecipitation specificity is high , the methylation chip signal is clear , and the positive control signal is clear .

( 2 ) In the common gene of the n - hexane exposure group , the demethylation gene was more than the hypermethylated gene .

( 3 ) Gene annotation indicates that the effect of n - hexane is mainly related to cellular process , metabolic process , intracellular gene and phosphotransferase activity gene .

( 4 ) Three DAVID cluster results of cell death , cell growth and hormone regulation showed that the methylation patterns of the control and 500 ppm groups were relatively close , and the methylation patterns of 2500 and 12500 ppm groups were relatively close .

( 5 ) The results of the signal pathway analysis showed that the apoptosis promoting gene in the apoptotic pathway was low methylation in the promoter region of the promoter region of the promoter region , and the apoptosis promoted the methylation of the promoter region of NFKBIA , Bid , Casp7 and Dffb in the 500 ppm group .

( 3 ) Study on the methylation of the gene promoter involved in the secretion of hormone in the ovary granular cells of F1 generation rats by n - hexane exposure during pregnancy

Method :

The methylation peak and the probe site of the promoter region of the gene related to the hormone synthesis in the MeDIP - Chip chip were determined . The methylation status of the promoter region of the hormone synthesis related gene was verified by the MS - MS method , and the mRNA expression level of the screening gene was determined by the RealtimePCR method .

Results :

( 1 ) The methylation peak of the promoter region of the gene related to hormone synthesis was measured : Star gene , the degree of methylation of 500 ppm group was lower than that of the control group , while the 12500ppm group was significantly higher than that of the control group ( p < 0.05 ) ;
The methylation degree of Cyp11a1 gene , 2500ppm and 12500ppm group was higher than that of control group ( p < 0.05 ) .
The methylation degree of Cyp17a1 gene , 2500ppm and 12500ppm group was higher than that of control group ( p < 0.05 ) .
The methylation degree of Hsd3b gene and 12500ppm group was significantly higher than that of control group ( p < 0.05 ) .

( 2 ) the degree of methylation of the local region of the Star gene promoter from high to low was 12500ppm2500ppmcontrol500ppm100 ppm ;
the degree of methylation of the partial region of the promoter of the Cyp11a1 gene ranges from 12500ppm to 2500ppm control500ppm and 100 ppm ;
The degree of methylation of the partial region of the Cyp17a1 promoter ranged from 1250 ppm to 2500 ppm control500 ppm and 100 ppm ;
The degree of methylation of the local region gene of the Hsd3b promoter ranged from 12500 ppm to 2500 ppm and Control100 ppm and 500 ppm .

( 3 ) RT - PCR results showed that the expression of Star , Cyp11a1 , Cyp17a1 and Hsd3bmRNA was significantly different from that of the control group , which was as follows : Compared with the control group , the expression level of the Star gene and the 500ppm group was significantly increased ( p < 0.05 ) , and the expression level in the 12500ppm group was significantly decreased ( p < 0.05 ) ;
The expression of Cyp11a1 gene and 500 ppm group was significantly increased ( p < 0.05 ) , and the expression level of Cyp11a1 gene was significantly decreased ( p < 0.05 ) .
The Cyp17a1 gene , 500 ppm group increased significantly ( p0.05 ) , 2500 ppm and 12500 ppm decreased significantly ( p < 0.05 ) .
Hsd3b gene , 12500ppm group decreased significantly ( p < 0.05 ) .

Conclusion :

Based on the above results , the following conclusions can be drawn :

1 . High - concentration n - hexane exposure during pregnancy has obvious embryotoxicity and can affect the ovarian development and hormone secretion function of F1 generation female rats .

2 . Normal hexane exposure during pregnancy alters the methylation status of the DNA promoter region of the ovary granular cells in the ovary , and the high - dose n - hexane exposure ( 2500 and 12500ppm ) can significantly change the methylation state of the apoptosis gene and the hormone synthesis gene of the granular cell .

3 . After the exposure of n - hexane in pregnancy , the methylation status of the local region of the promoter of the hormone synthesis related genes Star , Cyp11a1 , Cyp17a1 and Hsd3b in the ovary granular cells changed with the level expression of the corresponding gene mRNA , and the change in the secretion ability of the sex hormone may have a certain relationship .

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

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