【作者】 李宏；

【导师】 张文昌；

【作者基本信息】 福建医科大学 ， 药理学， 2013， 博士

【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（p<0.05）；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（p>0.05），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（p>0.05）。（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（p<0.05），500ppm和2500ppm组动情期显著延长（p<0.05），12500ppm组动情间期显著缩短（p<0.05）。（4）卵泡数目构成比：12500ppm组的次级卵泡比例显著低于对照组（p<0.05），闭锁卵泡比例显著高于对照组（p<0.05）。（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（p<0.05）；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（p<0.05）。（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，DNeasyBlood&TissueKit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PeakScore>2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（p<0.05）；Cyp11a1基因，2500ppm和12500ppm组的甲基化程度明显高于对照组（p<0.05）；Cyp17a1基因，2500ppm和12500ppm组的甲基化程度明显高于对照组（p<0.05）；Hsd3b基因，12500ppm组的甲基化程度显著高于对照组（p<0.05）。（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：12500ppm>2500ppm>control>500ppm>100ppm；Cyp11a1基因启动子局部区域甲基化程度从高到低排列为：12500ppm>2500ppm>control>500ppm和100ppm；Cyp17a1启动子局部区域甲基化程度从高到低排列为：12500ppm和2500ppm>control>500ppm和100ppm；Hsd3b启动子局部区域基因甲基化程度从高到低排列为：12500ppm>2500ppm和Control>100ppm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（p<0.05），12500ppm组表达量显著下降（p<0.05）；Cyp11a1基因，500ppm组表达量显著增高（p<0.05），12500ppm表达量显著下降（p<0.05）；Cyp17a1基因，500ppm组显著增高（p<0.05），2500ppm和12500ppm显著下降（p<0.05）；Hsd3b基因，12500ppm组显著下降（p<0.05）。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。更多还原

【Abstract】 N-hexane is a kind of solvent that widely used in food making, medicine production, and the organics synthesis. As it is volatile at the room temperature, the inhalation is the most frequent way in the occupational exposure. N-hexane is previously considered to be a low or micro-poisonous chemical so as to be widely used in various industries, the "Poisonous Apple" incident in2008had drawn public attentions, however, recent studies have shown that n-hexane and its metabolites had serious effects on female endocrine and reproductive system. In addition, The fundamental dates of toxicology had indicated that n-hexane exposure during gestation might induce offspring dysplasia and no gene mutations induced by were observed. As the above, we had established a model of pregnancy n-hexane exposure in order to detect the role of DNA methylation in the ovary development and ovarian granulosa cell functions in the adult offspring.Objective:To detect the impact on the development of reproductive system in the female offspring after gestational n-hexane exposure;another purpose was to investigate the methylation status of genome promoter, differential gene ontology, and methylation patterns in the ovarian granulosa cells of F1female rats after n-hexane exposure during pregnancy. And to explore the relationship between promoter methylation status, mRNA levels of the hormone synthesis genes, and the hormond levels in the ovarian granulosa cells of F1female rats after n-hexane exposure during pregnancy.Methods:1. Clean grade Wistar rats, whose body weights ranged from210-230g for females and300-320g for males, were caged. Each group contained5pregnant animals. F0pregnant rats inhaled n-hexane on gestational days1-20(4h per day) at doses of0,100,500,2500, and12500ppm. In the F1rats the sex ratio at birth, body weight growth, vaginal opening time, and estrous cycle were observed. Tissue sections were cut from each rat’s ovary with a rotary microtome, and then consecutively stained with hematoxylin and eosin (H&E). After these, the proportion of each stage follicles was calculated and pathologic changes were detected. The ovarian granulosa cells of the female F1generation was isolated and cultured for4h, then the progesterone and estradiol in the supernatant of the culture solution were measured using Chemiluminescent enzyme immunoassay (CLEIA)2. Genome DNA (gDNA) of ovarian granulosa cells in F1generation females was extracted using DNeasy Blood&Tissue Kit, The gDNA of each sample was sonicated and immunoprecipitated with5-mC antibody. Later the immunoprecipitated gDNA was hybridized with Nimblegen385Kpromoter Plus CpG Island Arrays. The differential methylation genes were defined as Peak Score>2compared with control. Several methods were used to analyze the differential genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering, and KEGG pathway analysis.3. The differential methylation peak (compared with control) in the promoter of hormone synthesis genes were determined using Signalmap software, afterwards the methylation status were verified by MS-HRM method. The mRNA levels of corresponding genes were determined using RT-PCR. Results:1. The live births of12500ppm group were significantly fewer than any other group, the male/female ratio showed a downtrend as the exposure dose increased. There were no differences between the control and n-hexane exposure group in body weight growth, however, n-hexane exposure group seemed to show a slower increase of body weights after PD36. There were also no differences between the control and n-hexane exposure group in virginal opening time. Compared with the control, the preoestrus durations were significantly longer in100,500, and2500ppm groups; the estrus durations of500and2500ppm groups were longer than that of the control. The diestrus duration and of12500ppm groups were shorter than the control. The atretic follicles proportion in12500ppm was lower than the control. Estradiol levels of2,500ppm and12,500ppm group were significantly lower than that of the control. Compared with the control, progesterone levels in100ppm and500ppm exposure group were significantly higher than that of the control, but progesterone levels in12,500ppm were significantly lower.2. The number of shared demethylated genes was higher than that of methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the500ppm and control groups were different from those of the2,500and12,500ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1, and Srd5a1promoters were hypermethylated in the n-hexane-exposed groups.3. promoter methylation of the genes related with hormone secretion in the ovarian granulosa cells of rat offspring:(1) Star, Cyp11a1, Cyp17a1, and Hsd3b genes showed differential promoter methylation of after n-hexane exposure. The specific as follows: In500ppm group, compared with control promoter methylation degrees of Star, Cyp11a1and Cyp17a1were lower, meanwhile those of Hsd3b showed no difference. In2500ppm group, compared with control, promoter methylation degrees of Cyp11a1and Cyp17a1were higher, meanwhile those of Star and Hsd3b showed no difference. In12500ppm group, compared with control, promoter methylation degrees of all the four genes were higher than those of the control.(2) Using the MS-HRM, the methylation degrees in local region of the Star gene promoter were as below:12500ppm>2500ppm> control>500ppm>100ppm; the methylation degrees in local region of the Cyp11al gene promoter were as below:25%>12500ppm>2500ppm> control>500ppm和100ppm; The methylation degrees in local region of the Cyp17al gene promoter were as below:12500and2500ppm> control>500and100ppm; the methylation degrees in local region of the Hsd3b gene promoter were as below:12500ppm>2500ppm and Control>100and500ppm. In total, results of MS-HRM were consistent with those of the MeDIP-CHIP.(3) Star, Cyp11a1, Cyp17a1, and Hsd3b showed differential mRNA levels after n-hexane exposure. The specific as follows:in Star and Cypllalgene expression, compared with control, the mRNA levels of12500ppm group were significantly lower, but those of500ppm group were significantly higher; in cyp17al gene expression, the mRNA levels of2500and12500ppm group were significantly lower than those of the control; in Hsd3b gene expression, the mRNA levels of12500ppm group were significantly lower than those of the control.Conclusions:1. N-hexane exposure during pregnancy had obvious embryo toxicity, and interfered with ovarian developments and functions in adult F1female offspring.2. N-hexane exposure during pregnancy could alter the methylation status of the promoter in the offspring ovarian granulosa cells, especially high doses of n-hexane exposure (2500and12500ppm) significant changed gene promoter methylation of cell apoptosis and hormone synthesis genes.3. After n-hexane exposure in gestation, the relationships between sex hormone secretion ability, the promoter methylation and mRNA levels alteration of genes Star, Cyp11a1, Cyp17al and Hsd3b in F1ovarian granulosa cells might exist.更多还原