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基于转录组测序探究BaP暴露对孕早期卵巢黄体衰老的影响
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Abstract:
目的：通过转录组测序技术分析孕早期BaP暴露对卵巢黄体的影响。方法：利用雌性SD大鼠构建BaP染毒孕鼠模型，BaP组孕鼠自怀孕第一天(d1)每日晨灌胃给予0.2 mg/(kg/d) BaP，对照组则给予等体积的玉米油，收集d7大鼠卵巢组织。同时利用0.5 μmol/L BaP处理人卵巢颗粒细胞KGN构建细胞模型。利用转录组测序(RNA-seq)技术检测对照组与BaP组卵巢黄体组织中的差异表达基因(DEGs)，并进一步对筛选的DEGs进行生物信息学分析和分子功能验证。结果：RNA-seq共筛选出300个差异表达基因，其中上调基因86个，下调基因214个。对DEGs进行GO功能注释和KEGG富集分析，结果显示BaP暴露可能通过细胞衰老信号通路损伤孕早期卵巢功能。β-半乳糖苷酶染色结果显示，与对照组相比，BaP处理组β-半乳糖苷酶活性显著增加；Western blot、免疫组化和免疫荧光结果显示，BaP暴露下调CDK4、CyclinD1的表达，同时上调p53、p21和p16的蛋白水平。结论：BaP暴露诱导卵巢颗粒细胞衰老，进而损伤孕早期大鼠卵巢黄体功能。
Objective: The effect of early pregnancy exposure to BaP on the ovarian corpus luteum was analysed by RNA-Seq. Methods: A BaP-contaminated pregnant mice model was constructed using female SD rats. The pregnant rats in the BaP group were given 0.2 mg/(kg/d) BaP by daily morning gavage up from the first day of pregnancy (d1), while the control group was given an equal volume of maize oil, and ovary tissues were collected from d7 rats. Treating human ovarian granulosa cells KGN with 0.5 μmol/L BaP to establish the cell models. The transcriptome sequencing (RNA-seq) technology was used to detect differentially expressed genes (DEGs) in ovarian corpus luteum tissues of the control and BaP groups, and the screened DEGs were further subjected to bioinformatics analyses and molecular functional validation. Results: A total of 300 DEGs were screened by RNA-seq, including 86 up-regulated genes and 214 down-regulated genes. Gene Ontology (GO) functional annotation and KEGG enrichment analyses of DEGs showed that BaP exposure may impair ovarian function in early pregnancy through cellular senescence signalling pathways. The results of β-galactosidase staining showed a significant increase in β-galactosidase activity in the BaP-treated group compared to the control group. Western blot, IHC and IF results revealed that BaP exposure down-regulated the expression of CDK4 and CyclinD1, while up-regulating the protein levels of p53, p21 and p16. Conclusion: BaP exposure might induce ovarian granulosa cell senescence, which leads to impair ovarian luteal function in early pregnancy rats.
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