【作者】 申林；

【导师】 章汉旺；

【作者基本信息】 华中科技大学 ， 妇产科学， 2024， 硕士

【摘要】 目的:环磷烯胺(cyclophosphamide,CTX)是临床广泛应用的化疗药物,会导致卵巢损伤,包括卵母细胞的数量和质量下降。β-烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)是烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)的前体,本研究拟探究腹腔注射NMN能否对抗CTX造成的卵巢损伤。方法:本研究将8周雌性C57小鼠随机分为3组:对照组(Control组)、化疗损伤组(CTX组)、NAD+保护组(NMN+CTX组)。CTX组和NMN+CTX组接受腹腔注射CTX 150 mg/kg。NMN+CTX组每天腹腔注射200 mg/kg的NMN,CTX组和对照组注射等体积生理盐水。14天后处死小鼠,评估各组卵巢、卵母细胞及胚胎共三个层面的生育力情况,包括:1)卵巢的储备情况、内分泌功能及NAD(H)含量;2)促排卵得到的成熟卵母细胞质量;3)卵母细胞体外受精过程中胚胎的发育潜能。此外,对卵巢和卵母细胞测序,寻找潜在机制,分析并验证两者转录组变化。结果:本研究发现CTX组卵巢内NAD+含量显著降低,NMN+CTX组卵巢内NAD+的含量明显提高。NMN+CTX组小鼠卵巢的储备情况、内分泌功能及NAD+含量得到改善;卵母细胞纺锤体、胚胎发育潜能损害程度减轻;卵母细胞和囊胚的氧化应激和DNA损伤水平降低。卵巢和卵母细胞测序结果提示氧化应激应答基因和DNA损伤应答基因受到CTX和NMN的强烈影响,并可能发挥潜在作用。结论:NMN可提高卵巢NAD+水平,改善CTX对卵巢储备和内分泌功能、卵母细胞质量及胚胎发育潜能的损害,并降低CTX所致卵母细胞和胚胎氧化应激和DNA损伤,减少卵母细胞和胚胎凋亡。第一部分:NAD+在化疗过程中对卵巢功能的保护作用及其相关机制目的:探究CTX和补充NMN对卵巢的卵泡储备、内分泌功能和NAD(H)含量的影响;以及卵巢内受CTX和NMN影响的通路和基因变化。方法:该部分将8周雌性C57小鼠随机分为3组:健康对照组(Control组)、化疗损伤组(CTX组)、NAD+保护组(NMN+CTX组)。CTX组和NMN+CTX组接受腹腔注射环磷酰胺150 mg/kg。NMN+CTX组造模期间每天腹腔注射200 mg/kg的NMN,CTX组和对照组注射等体积生理盐水。14天后处死小鼠,取卵巢固定后切片染色,计数各时期卵巢卵泡;取小鼠眼球静脉血血清,测量小鼠血清中抗缪勒管激素(anti-Müllerian hormone,AMH)和促卵泡激素(follicle-stimulating hormone,FSH)的含量;使用NAD(H)检测试剂盒检测单位质量卵巢内NAD(H)的含量;将新鲜取得的卵巢行二代转录组测序,对质检合格的测序数据进行分析并验证。结果:NMN+CTX组卵巢的CTX毒性减低。与CTX组相比,NMN+CTX组始基卵泡和窦卵泡数量上升,异常的始基卵泡和窦卵泡比例恢复,卵巢指数升高、NAD+含量及NAD(H)含量上升。通过卵巢转录组测序发现,CTX使得卵巢氧化应激和DNA损伤应答基因表达发生改变,其中RNA结合基序蛋白(RNA binding motif protein 47,Rbm47)和BTG3相关核蛋白(BTG3 associated nuclear protein,Banp)的表达受到明显抑制,补充NMN使得卵巢Rbm47和Banp表达上升。结论:补充NMN可显著减少CTX对卵巢的毒性作用,改善CTX对卵巢储备、功能和卵巢指数和NAD+含量的损害,并改善CTX对卵巢内氧化应激和DNA损伤相关基因Rbm47和Banp的表达抑制。第二部分:NAD+在化疗中对小鼠卵母细胞和胚胎质量的改善作用及其相关机制目的:探究CTX和NAD+对卵母细胞和胚胎的影响,即补充NMN能否抵抗CTX对卵母细胞及胚胎的损害,以及补充NMN发挥作用的机制。方法:该部分的分组和处理方法与第一部分相同。造模期间对各组小鼠进行促排卵后,在第13天处死小鼠获取卵母细胞,记录每组每只小鼠的获卵数;对卵母细胞染色评估纺锤体形态、DNA损伤、氧化应激水平,并对其进行体外受精后观测其发育潜能,以及所形成囊胚的DNA损伤、氧化应激水平。同时,将所得成熟的卵母细胞行单细胞转录组测序,探究潜在应答机制,尤其是氧化应激和DNA损伤应答基因的作用。结果:腹腔注射NMN补充NAD+的含量可显著改善CTX对卵母细胞的损害,包括改善卵母细胞异常纺锤体和细胞核形态、降低DNA损伤程度与氧化应激水平。此外,体外受精结果显示NAD+的补充可改善CTX对胚胎发育潜能的影响,提高胚胎的受精率及成囊率,降低胚胎异常的氧化应激和DNA损伤水平。卵母细胞测序数据表明,在CTX损伤和NMN保护卵母细胞和胚胎过程中,氧化应激和DNA损伤应答相关基因和通路显著变化,其中血清/糖皮质激素调节激酶(serum/glucocorticoid regulated kinase 1,Sgk1)和谷氨酰胺转移酶A(proteinglutamine gamma-glutamyltransferase A,F13a1)的表达受到CTX抑制,NMN的补充可恢复Sgk1和F13a1的表达。结论:腹腔注射NMN可显著改善CTX对卵母细胞质量及胚胎发育潜能的影响,并能降低囊胚的氧化应激和DNA损伤水平。对卵母细胞行单细胞测序分析并验证,发现潜在的靶点为Sgk1。更多还原

【Abstract】 Objective: Cyclophosphamide(CTX),a widely used chemotherapeutic drug in clinical practice,causes ovarian damage,including a reduction in the number and quality of oocytes.β-nicotinamide mononucleotide(NMN)is the precursor of nicotinamide adenine dinucleotide(NAD+),and the present study was designed to investigate whether intraperitoneal injection of NMN could counteract the ovarian damage caused by CTX.Methods: In this study,8-week-old female C57 mice were randomly divided into 3 groups:control group,chemotherapeutic injury group(CTX group),and NAD+ protection group(NMN+CTX group).The CTX and NMN+CTX groups received intraperitoneal injections of CTX at 150 mg/kg.The NMN+CTX group received intraperitoneal injections of NMN at 200 mg/kg per day,and CTX and control groups were injected with equal volumes of saline.After 14 days,the mice were sacrificed and the fertility was assessed at three levels:1)ovarian reserve,endocrine function,and NAD(H)content;2)quality of mature oocytes obtained by ovulation promotion;and 3)developmental potential of embryos during in vitro fertilization of oocytes.In addition,ovaries and oocytes were sequenced to look for potential mechanisms to analyze and validate transcriptomic changes in both.Results: In this study,it was found that the content of NAD+ in the ovary was significantly reduced in the CTX group,and the content of NAD+ in the ovary was significantly increased in the NMN+CTX group.In the NMN+CTX group,the ovarian reserve,endocrine function and NAD+ content of mice were improved;the damage to the oocyte spindle and embryonic developmental potential was reduced;and the levels of oxidative stress and DNA damage in oocytes and blastocysts were reduced.The ovarian and oocyte transcriptomes suggested that oxidative stress response genes and DNA damage response genes are strongly influenced by CTX and NMN and may play a potential role.Conclusions: NMN increased ovarian NAD+ levels,ameliorated CTX damage to ovarian reserve and endocrine function,oocyte quality and embryonic developmental potential,and reduced CTX-induced oxidative stress and DNA damage in oocytes and embryos,and reduced oocyte and embryo apoptosis.Part Ⅰ: Protective effect of NAD+ on ovarian function during chemotherapy and its associated mechanismsObjective: To investigate the effects of CTX and NMN supplementation on follicular reserve,endocrine function and NAD(H)content of the ovary,as well as the pathways and genetic changes in the ovary that are affected by CTX and NMN.Methods: In this section,8-week-old female C57 mice were randomly divided into 3 groups: control group,chemotherapy injury group(CTX group),and NAD+ protection group(NMN+CTX group).The CTX and NMN+CTX groups received intraperitoneal injections of CTX at 150 mg/kg.The NMN+CTX group received intraperitoneal injections of NMN at 200 mg/kg per day,and CTX and control groups were injected with equal volumes of saline.After 14 days,the mice were sacrificed,the ovaries were taken,fixed,sectioned and stained,then the ovarian follicles were counted in each period;the serum was obtained from the venous blood of the eyes of the mice,and the serum was measured for the anti-müllerian hormone(AMH)and follicle-stimulating hormone(FSH)in the mice.The NAD(H)test kit was used to measure the NAD(H)content per unit mass of ovary;the freshly obtained ovaries were subjected to second-generation transcriptome sequencing,and the qualified sequencing data were analyzed and validated.Results: CTX toxicity was reduced in the ovaries of the NMN+CTX group.Compared with the CTX group,the number of primordial follicle and antral follicle increased,the proportion of abnormal primordial follicle and antral follicles was restored,and the ovarian index was elevated,and the NAD+ and NAD(H)content increased in the NMN+CTX group.Ovarian transcriptome revealed that CTX altered the expression of oxidative stress and DNA damage response genes in the ovary,in which the expression of RNA binding motif protein 47(Rbm47)and BTG3 associated nuclear protein(Banp)was significantly suppressed.expression was significantly suppressed,and NMN supplementation resulted in increased expression of Rbm47 and Banp in the ovary.Conclusions: NMN supplementation significantly reduced the toxic effects of CTX on the ovary,ameliorated CTX damage to ovarian reserve,function and ovarian index and NAD+ content,and deregulated CTX inhibition of the expression of genes related to oxidative stress and DNA damage(Rbm47 and Banp)in the ovary.Part Ⅱ: Improvement of oocyte and embryo quality in mice by NAD+ in chemotherapy and its associated mechanismsObjective: To investigate the effects of CTX and NAD+ on oocytes and embryos,i.e.whether NMN supplementation can counteract the damage caused by CTX to oocytes and embryos,and the mechanisms by which NMN supplementation works.Methods: This part was grouped and treated in the same way as Part I.Each group of mice was controlled ovarian hyperstimulation on day 10,and the mice were killed 14-16 h later to obtain oocytes on day13.The number of oocytes obtained from each mouse in each group was recorded;the oocytes were stained to assess the spindle morphology,DNA damage and oxidative stress levels,and the developmental potential of the oocytes was observed after in vitro fertilization,as well as the DNA damage and oxidative stress levels of the resulting blastocysts.At the same time,the mature MII oocytes were subjected to single-cell transcriptome sequencing to investigate the underlying response mechanisms,especially the roles of oxidative stress and DNA damage response genes.Results: Intraperitoneal injection of NMN significantly ameliorated the effects of CTX on oocyte damage,including improvement of abnormal spindle and nucleus morphology,and reduction of DNA damage and oxidative stress in oocytes.In addition,in vitro fertilization results showed that NAD+ supplementation ameliorated the effects of CTX on the developmental potential of the embryo,increased the fertilization and blastocyst rate of the embryo,and reduced the abnormal levels of oxidative stress and DNA damage in the embryo.Oocyte sequencing data showed significant changes in genes and pathways related to oxidative stress and DNA damage response during CTX injury and NMN protection of oocytes and embryos,with significant changes in Serum/Glucocorticoid Regulated Kinase 1(Sgk1)and Protein-Glutamine Gamma-Glutamyltransferase A(F13a1)expression was inhibited by CTX,and NMN supplementation restored Sgk1 and F13a1 expression.Conclusions: Intraperitoneal injection of NMN significantly ameliorated the effects of CTX on oocyte quality and embryonic developmental potential,and reduced the levels of oxidative stress and DNA damage in blastocysts.Single-cell sequencing analysis and validation of oocytes revealed that the potential target was Sgk1.更多还原