先天性心脏病（CHD）是婴儿因出生缺陷死亡的最常见原因，在我国出生缺陷中位居首位。先天性心脏病的主要特征是胚胎发育过程中心脏或大血管的大体结构异常，包括房间隔缺损、室间隔缺损、主动脉干永存和心肌致密化不全等，它是一种多因素的复杂疾病，由遗传因素、环境因素以及遗传和环境交互作用引起。心肌致密化不全是一种直接影响心肌组织内壁的先天性病变，导致心脏内壁血液充盈形成小梁、心膜变薄和心肌细胞增殖活性降低等。五氯硝基苯（PCNB）是一类有机氯农药，广泛分布在田地和草场等区域。我们实验室前期研究发现小鼠孕期灌胃暴露PCNB诱导胎鼠心肌致密化不全样病理表型，通过转录组测序筛选对照和PCNB处理组E17.5   胎鼠心脏差异表达基因，发现PCNB心脏毒性机制与PCNB下调Ndc80复合物成员Hec1的表达有关，导致染色体排列和分离异常，抑制心肌细胞有丝分裂和增殖。此外，我们实验室前期报道PCNB能改变青春前期大鼠卵巢甾体激素的合成，加速原始卵泡的发育。本论文基于前期研究，探究了PCNB下调心脏Hec1表达及影响卵泡发育的分子机制。

我们以H9c2和HL-1心肌细胞作为实验材料，用不同浓度PCNB进行处理， Western blot检测结果表明PCNB暴露显著降低Hec1蛋白表达水平。为了探究PCNB下调Hec1的分子机制，我们克隆并鉴定了Hec1的启动子区，通过Jaspar和hTF target软件预测了Hec1启动子区潜在的转录因子，结合PCNB处理组E17.5胎鼠心脏获得的差异表达基因，候选压力响应型转录因子ATF3进行后续研究。利用对照和PCNB处理组心脏组织进行Real time PCR检测，证实PCNB显著下调心脏组织ATF3的转录水平。免疫组化结果显示，ATF3在PCNB处理组的E17.5胎鼠心脏组织中表达量明显减少。用不同浓度PCNB处理H9c2和HL-1细胞后，Western blot结果显示ATF3的蛋白表达水平显著降低。这些结果与PCNB下调心脏组织和体外心肌细胞的Hec1的表达一致。

为了探究ATF3与Hec1之间的调控关系，我们构建Myc-ATF3真核表达质粒，将空载体、Myc-ATF3表达质粒和Hec1启动子报告质粒分别共转染HEK293ET细胞，双荧光素酶报告基因实验检测发现ATF3能激活Hec1启动子。我们将空载体和Myc-ATF3表达质粒分别转染HEK293ET和H9c2细胞，Real-time PCR检测表明ATF3过表达显著升高Hec1的mRNA水平，而特异性靶向ATF3的siRNA分别转染HEK293ET和H9c2细胞后都显著降低Hec1的mRNA水平。Western blot结果显示ATF3过表达显著升高Hec1的蛋白水平，而ATF3敲低显著降低Hec1的蛋白水平。我们进一步通过Jaspar软件预测分析ATF3结合在Hec1启动子的位点，发现Hec1启动子区-26/-19位点存在ATF3可能识别的ATF/CRE元件，构建针对-26/-19区域的缺失和点突变报告质粒，进行双荧光素酶报告基因实验，结果显示-26/-19元件的缺失突变和点突变都显著降低了ATF3对Hec1启动子的转录激活作用，表明Hec1启动子区的ATF/CRE元件介导了ATF3的转录激活作用。染色质免疫沉淀实验结果显示ATF3能结合含ATF/CRE元件的区域。这些研究表明ATF3能转录激活Hec1的表达。

随后我们分析了ATF3在PCNB抑制心肌细胞增殖中的作用，用EdU和H3组蛋白在第10位丝氨酸磷酸化修饰（pH3）作为标志物进行免疫荧光染色，分别检测H9c2细胞增殖和有丝分裂情况。结果显示ATF3过表达援救了PCNB对心肌细胞增殖和有丝分裂的抑制作用，而ATF3敲低加剧了PCNB抑制心肌细胞的增殖和有丝分裂。纺锤体标志物β-tubulin免疫荧光染色显示ATF3过表达援救了PCNB诱导的纺锤体形态和染色体分离异常，而ATF3敲低加剧了纺锤体形态和染色体分离异常。

结合Hec1在体外心肌细胞增殖中的作用研究，为了进一步探索Hec1对心脏发育的影响，我们利用CRISPR/Cas9技术构建了Hec1心脏特异性条件敲除小鼠，基因型鉴定结果显示得到了野生型和杂合型的小鼠，未得到纯合子基因型小鼠，提示条件性敲除Hec1的纯合子小鼠可能因为Hec1缺失引起心脏发育过程中心肌细胞增殖异常导致胚胎致死。

PCNB影响大鼠卵泡发育的分子机制，我们将青春前期大鼠进行PCNB灌胃一周后，卵巢组织HE染色观察并统计大鼠卵巢各级卵泡数目，发现PCNB处理组原始卵泡显著减少，生长卵泡显著增加，确定了PCNB加速大鼠卵泡发育。随后通过RNA-Seq 测序筛选对照组和PCNB处理组卵巢组织差异表达基因，共筛选出 261 个差异表达基因，其中上调基因有 62个，下调基因有199 个。GO分析显示差异表达基因主要富集在细胞分化、细胞内信号转导、ATP结合的微管装配等。KEGG富集分析提示在卵泡发育调控中扮演重要作用的PI3K-AKT信号通路富集的差异基因数目较多。Western Blot结果显示PCNB处理组AKT磷酸化水平显著增加，而负调AKT信号通路的PTEN基因表达量显著减少。这些结果表明PCNB激活卵巢组织AKT信号通路。

ATF3能转录激活Hec1的表达，调控心肌细胞有丝分裂过程中染色体分离，ATF3-Hec1轴下调部分介导PCNB抑制心肌细胞增殖和有丝分裂，这些研究提示PCNB可能部分通过下调ATF3-Hec1轴引起心肌致密化不全样表型。此外，我们的研究结果初步表明PCNB加速大鼠原始卵泡发育与PI3K-AKT信号通路激活相关。

Congenital heart disease (CHD) is the most common cause of infant death due to birth defects, ranking first among birth defects in China.  Congenital heart disease is characterized by gross structural abnormalities of the heart or large blood vessels during embryonic development, including atrial septal defect, ventricular septal defect, aortic trunk perpetuation and myocardial incompleteness, etc. It is a multi-factor complex disease caused by genetic factors, environmental factors and genetic and environmental interaction. Noncompaction of ventricular myocardium is a congenital disease that directly affects the inner wall of the cardiac tissue, leading to the formation of trabecular blood filling in the inner wall of the heart, thinning of the cardiac membrane and reduced proliferation activity of cardiac cells.  Pentachloronitrobenzene (PCNB) is a kind of organochlorine pesticide, which is widely distributed in fields and grasslands.  Previous studies in our laboratory found that mice exposed to PCNB during pregnancy induced incomplete myocardial densification phenotype in fetal mice. Transcriptome sequencing was used to screen differentially expressed genes in E17.5 fetal rat hearts of control and PCNB treatment groups, and the cardiotoxicity mechanism of PCNB was found to be related to PCNB down-regulating the expression of Hec1, a member of Ndc80 complex.  Lead to abnormal chromosome arrangement and separation, inhibit myocardial cell mitosis and proliferation.  In addition, our laboratory previously reported that PCNB can change the synthesis of steroid hormones in the ovary of prepubertal rats and accelerate the development of primordial follicles.  Based on previous studies, this paper explored the molecular mechanism of PCNB down-regulating cardiac Hec1 expression and affecting follicular development.  

H9c2 and HL-1 cardiomyocytes were used as experimental materials and treated with different concentrations of PCNB. Western blot analysis showed that PCNB exposure significantly reduced protein expression level of Hec1.  In order to explore the molecular mechanism of PCNB down-regulating Hec1, we cloned and identified the Hec1 promoter region, predicted the potential transcription factors of Hec1 promoter region by Jaspar and hTF Target software, combined with the differentially expressed genes obtained by PCNB treatment of E17.5 fetal rat heart.  The candidate stress response transcription factor ATF3 was further studied.  Real-time PCR was performed on E17.5 heart tissue by intragastric administration of control and PCNB to confirm that PCNB significantly down-regulated the transcription level of ATF3 in heart tissue.  Immunohistochemical results showed that the expression of ATF3 in E17.5 mice heart tissues of PCNB treated group was significantly reduced.  After H9c2 and HL-1 cells were treated with different concentrations of PCNB, Western blot results showed that the protein expression level of ATF3 was significantly decreased.  These results are consistent with PCNB down-regulating Hec1 expression in cardiac tissue and cardiomyocytes in vitro.  

In order to explore the regulatory relationship between ATF3 and Hec1, we constructed Myc-ATF3 eukaryotic expression plasmid, and co-transfected HEK293ET cells with empty vector, Myc-ATF3 expression plasmid and Hec1 promoter reporter plasmid, respectively. Double luciferase reporter gene assay showed that ATF3 could activate Hec1 promoter.  Empty vector and Myc-ATF3 expression plasmid were transfected into HEK293ET and H9c2 cells respectively. Real-time PCR showed that ATF3 overexpression significantly increased mRNA level of Hec1.  The specific ATF3-targeting siRNA transfected into HEK293ET and H9c2 cells significantly reduced the mRNA level of Hec1.  Western blot results showed that ATF3 overexpression significantly increased Hec1 protein level, while ATF3 knockdown significantly decreased Hec1 protein level.  Jaspar software was further used to predict and analyze the binding site of ATF3 to Hec1 promoter, and it was found that there were ATF/CRE elements that could be recognized by ATF3 at the -26/-19 site of Hec1 promoter. The reporter plasmid for deletion and point mutation in the -26/-19 region was constructed and the double luciferase reporter experiment was conducted.  The results showed that both deletion mutation and point mutation of -26/-19 element significantly reduced the transcriptional activation of ATF3 on Hec1 promoter, suggesting that ATF/CRE element of Hec1 promoter mediated the transcriptional activation of ATF3.  Chromatin immunoprecipitation assay showed that ATF3 could bind to regions containing ATF/CRE elements.  These studies suggest that ATF3 can transcriptionally activate Hec1 expression.  

Subsequently, we analyzed the role of ATF3 in inhibiting proliferation of cardiac myocytes by PCNB, and detected proliferation and mitosis of H9c2 cells by EdU and pH3 immunofluorescence staining, respectively.  The results showed that overexpression of ATF3 rescued the inhibitory effect of PCNB on proliferation and mitosis of cardiomyocytes, while ATF3 knockdown intensified the inhibitory effect of PCNB on proliferation and mitosis of cardiomyocytes.  Immunofluorescence staining of β-tubulin, a spindle marker, showed that ATF3 overexpression rescued the spindle morphology and chromosome division abnormalities induced by PCNB, while ATF3 knockdown exacerbated the spindle morphology and chromosome division abnormalities.  

Combined with the studies on the role of Hec1 in the proliferation of cardiomyocytes in vitro, in order to further explore the effect of Hec1 on cardiac development, we constructed Hec1 heart-specific conditional knockout mice using CRISPR/Cas9 technology, and genotypic identification results showed that the wild-type and heterozygous mice were obtained，but homozygous mice were not obtained.  It is suggested that conditional Hec1 knockout homozygous mice genotype may cause abnormal proliferation of cardiac myocytes during heart development, resulting in embryonic death.  

In addition, in order to explore the molecular mechanism of PCNB effects in rats of follicular, we will be in the early stage of the youth PCNB in rats lavage after a week of ovarian tissue HE dyeing observation and statistics of rat ovarian follicle number at all levels, found PCNB treatment group had a significantly reduced, primordial follicle growth follicle significantly increased, and determines the PCNB accelerate follicle development in rats.  Subsequently, differentially expressed genes were screened by RNA-Seq in the control group and PCNB treatment group. A total of 261 differentially expressed genes were screened, including 62 up-regulated genes and 199 down-regulated genes.  GO analysis showed that differentially expressed genes were mainly enriched in cell differentiation, intracellular signal transduction, and ATP-bound microtubule assembly.  KEGG enrichment analysis suggested that there were a large number of differential genes in the enrichment of PI3K-AKT signaling pathway, which played an important role in the regulation of follicular development.  Western blot results showed that the phosphorylation level of AKT in PCNB treatment group was significantly increased, while the expression level of PTEN gene in negatively regulated AKT signaling pathway was significantly decreased.  These results suggest that PCNB activates the AKT signaling pathway in ovarian tissue.  

In summary, our study shows that ATF3 can transcriptional activate Hec1 expression and regulate spindle formation and chromosome division in cardiomyocyte mitosis. Down-regulation of ATF3-Hec1 axis partially mediates PCNB to inhibit cardiomyocyte proliferation and mitosis.  These studies suggest that PCNB may contribute to an incomplete myocardial densification phenotype, in part by down-regulating the ATF3-Hec1 axis.  In addition, our results preliminarily suggest that PCNB acceleration of primordial follicle development in rats is related to activation of PI3K-AKTsignaling pathway.