【作者】 乐芳；

【导师】 金帆；

【作者基本信息】 浙江大学 ， 妇产科学， 2013， 博士

【摘要】 第一部分体外受精对小鼠生长发育的影响及其肝脏和骨骼肌Igf2/H19的表达与表观遗传修饰研究目的：研究IVF小鼠的生长发育、肝脏及骨骼肌印记基因Igf2、H19、Igf2r与微小RNA (microRNA,miRNA)的表达、以及Igf2/H19差异甲基化区(differentially methylated region, DMR)的甲基化状况,阐明IVF对出生小鼠生长发育的短期及中长期效应和发生机制。材料和方法：1.利用C57BL/6J小鼠建立IVF及体内受精小鼠模型。比较IVF与体内受精子代的妊娠率、胎仔数、怀孕天数、性别比率及死亡率。2.以IVF子代为研究对象,以体内受精子代为对照组,自出生第1天起至第10周性成熟期测量各组小鼠体重变化；于3周(断乳期),10周(性成熟期),1.5年(老年期)取小鼠各器官,测量各组器官重量。3.通过定量PCR技术,检测出生、3周、10周及1.5年时期,肝脏及骨骼肌上印记基因Igf2、H19、Igf2r与miRNA-483的表达。4.利用亚硫酸氢盐PCR (bisulfite PCR, BSP)及克隆测序检测IVF小鼠的H19DMR、Igf2DMR2、Igf2r DMR2各位点的甲基化情况,再利用焦磷酸测序技术进行验证。5.通过western-blot技术,检测出生、3周、10周及1.5年时期,肝脏及骨骼肌上IGF2蛋白的表达。结果：1.建立IVF及体内受精小鼠各51只。两组小鼠在妊娠率、胎仔数、怀孕天数、性别比率及死亡率上差异无统计学意义。2.在出生时,IVF体重高于体内受精组,而发育至2周及3周时,IVF体重显著低于体内受精组,3周后两组的体重差异无统计学意义。器官重量,除去10周时IVF的脾脏重量低于体内受精组,两组小鼠的各器官重量在3周,10周,1.5年时差异均无统计学意义。3.IVF对出生后子代肝脏及骨骼肌Igf2/H19及miRNA-483表达的影响(1)肝脏上Igf2/H19及miRNA-483表达分析在出生及3周时,Igf2、H19与miRNA-483的表达在IVF组与体内受精组之间存在差异并有统计学意义。10周时,Igf2、H19及miRNA-483的表达在两组之间差异无统计学意义。但到老年时,Igf2、H19与miRNA-483的表达在IVF组与体内受精组之间又出现差异并有统计学意义。而在整个生长发育阶段,Igf2r的表达在两组之间差异均无统计学意义。(2)骨骼肌上Igf2/H19及miRNA-483表达分析在3周时,Igf2、H19与miRNA-483的表达在IVF组与体内受精组之间存在差异并有统计学意义。10周时,Igf2、H19及miRNA-483的表达在两组之间差异无统计学意义。但到老年时,Igf2、H19与miRNA-483的表达在IVF组与体内受精组之间又出现差异并有统计学意义。Igf2r的表达在3周及10周时,两组之间差异无统计学差异。但在老年时,Igf2r的表达在IVF组显著低于体内受精组。4.IVF对出生后子代肝脏及骨骼肌Igf2/H19DMR与Ig/2r DMR2甲基化的影响依据上述Igf2及H19的长期表达差异,选取H19DMR的12个甲基化位点,Igf2DMR2的13个甲基化位点进行BSP并克隆测序,结果发现：H19DMR的第6,7,8三个CpG位点,Ig/2DMR2的第4,5CpG位点在IVF组易出现甲基率的改变,因此,进一步对H19DMR第6到8CpG位点,Igf2r DMR2的第1到5CpG位点利用焦磷酸测序进行验证。同时选择Igf2r DMR2的4个CpG位点进行甲基化状况分析。(1)肝脏Igf2/H19DMR与Ig/2r DMR2的甲基化状态分析1)在出生时,H19DMR的甲基化率在IVF组与体内组之间存在差异并有统计学意义。在3周及10周时,H19DMR的甲基化率在两组之间差异无统计学意义。但到1.5年时,H19DMR的甲基化率在IVF组与体内受精组之间又出现差异并有统计学意义。2)在出生及3周时,H19DMR2的甲基化率在IVF组与体内组之间存在差异并有统计学意义。在10周时,Igf2DMR2的甲基化率在两组之间差异无统计学意义。但到1.5年时,Igf2DMR2的甲基化率在IVF组与体内受精组之间又出现差异并有统计学意义。3)在整个生长发育阶段,Igf2r DMR2的甲基化率在两组之间差异均无统计学意义。(2)骨骼肌Igf2/H19DMR与Igf2r DMR2的甲基化状态分析1)在3周时,H19DMR的第6到8三个CpG位点的甲基化率在IVF组均显著低于体内受精组。但在10周及1.5年时,H19DMR的甲基化率在两组之间差异无统计学意义。2)在3周时,Igf2DMR2的甲基化率在IVF组与体内组之间存在差异并有统计学意义。10周时,Igf2DMR2的甲基化率在两组之间差异无统计学意义。但到1.5年时,Igf2DMR2的甲基化率在IVF组与体内受精组之间又出现差异并有统计学意义。3)在3周及10周时,Igf2r DMR2的甲基化率在两组之间差异无统计学意义。但在1.5年时,Igf2r DMR2的4个CpG位点甲基化率在IVF组均高于体内受精组并有统计学意义。5.IVF对出生后子代肝脏及骨骼肌IGF2蛋白表达的影响在3周时期,无论是在肝脏还是骨骼肌,IGF2蛋白的表达在IVF组均显著低于体内受精组。其它生长发育阶段IGF2蛋白的表达在两组间差异无统计学差异。结论：1.IVF可影响子代的生长发育,但随着年龄的增长,这种改变逐渐修复。2.IVF不仅影响子代早期肝脏及骨骼肌Igf2、H19与miRNA-483的表达,还影响它们在老年时期的表达。3.IVF影响Igf2/H19的表达可能与它们的DMR区调控及miRNA-483相关,提示IVF可导致表观遗传学的改变。第二部分ART老年小鼠血脂代谢的改变及其机制研究目的：研究ART出生小鼠在老年时期的血脂代谢情况,探索ART对子代血脂改变的调控机制,明确ART致成年后疾病的发病风险,及阐明ART影响子代成年后健康的关键作用环节及致病时间窗。材料和方法：1.ART老年小鼠表型和血脂代谢检测(1)应用已建好的ART老年C57BL/6J小鼠模型包括：12只IVF小鼠、12只ICSI小鼠、10只IVM小鼠以及16只体内受精小鼠(每组小鼠雌雄比率相似),分析各组小鼠的表型和血脂代谢等状况。(2)通过水迷宫及恐惧学行为实验,分析各组ART老年小鼠的学习记忆能力。(3)利用血压检测仪,检测各组ART老年小鼠的血压及心率情况。(4)采用腹腔内葡萄糖注射实验,对ART各组小鼠在老年时期血糖及糖耐量进行检测。(5)利用生化分析仪,对ART各组老年小鼠血清进行生化分析包括血脂生化及肝肾生化。同时应用ELISA技术,检测各组老年小鼠血清胰岛素水平。(6)对各组ART老年小鼠的各器官进行称量,并通过HE染色对ART小鼠的不同器官进行病理学分析。2.机制学研究依据ART老年小鼠血脂代谢和器官病理改变的观察结果,选择脂类代谢关键调节通路：胰岛素诱导基因(insulin-induced gene, INSIG)、胆固醇调节元件结合蛋白(sterol regulatory element binding protein, SREBP)以及胆固醇调节元件结合蛋白裂解激活蛋白(SREBP cleavage-activating protein, SCAP),即INSIG-SCAP-SREBP为目标基因,选取肝脏和肺脏为靶组织,进行老年小鼠血脂代谢改变的发生机制研究。(1)ART老年小鼠INSIG-SCAP-SREBP基因表达分析利用荧光及探针定量RT-PCR检测ART老年小鼠肝脏和肺脏组织Insigl, Insig2, Scap, Srebf1, Srebf2, miR-33与miR-122的表达变化。(2)ART老年小鼠INSIG-SCAP-SREBP基因修饰分析应用BSP及焦磷酸测序技术检测ART老年小鼠肝脏和肺脏组织INSIG-SCAP-SREBP的CpG岛的甲基化状态。(3)ART老年小鼠INSIG-SCAP-SREBP蛋白表达分析通过western-blot及免疫组化等检测INSIG-SCAP-SREBP各蛋白在肝脏和肺脏组织的表达情况。(4) IVM的MⅡ卵母细胞INSIG-SCAP-SREBP表达分析利用定量RT-PCR及免疫荧光技术,检测IVM的MII卵母细胞INSIG-SCAP-SREBP基因及蛋白的表达变化。结果：1.ART老年子代的表型和血脂变化情况(1)各组ART老年小鼠与体内受精组相比较,学习记忆能力差异无统计学意义。(2)IVM老年小鼠血压显著高于IVF组、ICSI组及体内受精组,同时IVM组的心率显著高于体内受精组。IVF及ICSI组分别与体内组比较,在血压及心率上差异均无统计学意义。(3) ICSI组老年小鼠在进行糖耐量实验时,于葡萄糖注射30min后血糖水平显著低于体内受精组,差异具有统计学意义。(4)IVM及ICSI组的血清生化分析提示：与体内组比较,两组老年小鼠在血胆固醇、血脂蛋白(LDL-C, HDL-C及Apo-A)、血AST/ALT、血急性C反应蛋白存在差异并有统计学意义。另外,IVM组的血清胰导素水平显著高于体内受精组。(5) ICSI组及IVM组的肾脏和心脏重量均显著高于体内组及IVF组,差异具有统计学意义。另外,IVM组的胰腺及肝脏重量显著高于其它三组。(6)HE染色结果显示：IVM组老年小鼠的肾脏存在脂类改变；ICSI及IVF老年小鼠肺脏均存在炎性变化。2.在肝脏上,各组ART老年小鼠INSIG-SCAP-SREBP的表达变化依据上述血清生化分析结果,血脂改变主要发生于ICSI组及IVM组,因此肝脏组织血脂代谢研究以ART老年子代包括IVF组、ICSI组及IVM组为研究对象,以体内受精子代为对照。(1) mRNA水平：Insig1, Scap, Srebf1, Srebf2基因的表达在IVM及ICSI组的表达均显著高于IVF组及体内受精组(2-4倍,P<0.01)。另外,在IVM组及ICSI组,miR-33及miR-122表达均显著低于IVF及体内受精组。(2)DNA水平：Insig1, Scap, Srebf1, Srebf2的CpG岛的甲基化位点在IVM与ICSI组均与体内组存在甲基化率的差异,并有统计学意义。(3)蛋白水平：IVM组及ICSI组的SCAP蛋白表达显著高于IVF组及体内受精组,并有统计学意义。3.在肺脏上,各组ART老年小鼠INSIG-SCAP-SREBP的表达变化依据上述组织病理学结果,肺部炎症病变主要发生于IVF组及ICSI组,因此肺脏组织研究以ART老年子代包括IVF组及ICSI组为研究对象,以体内受精子代为对照；(1)mRNA水平：Insig1, Insig2, Scap, Srebf1, Srebf2基因的表达在ICSI及IVF组的表达均显著低于体内受精组,并有统计学意义。(2) DNA水平：Insig1, Scap, Srebf1, Srebf2的CpG岛的甲基化位点在IVF与ICSI组均与体内组存在甲基化率的差异,并有统计学意义。尤其是在病理肺脏上,各基因的甲基化率存在显著变化。(3)蛋白水平：western-blot及免疫组化结果显示,IVF及ICSI组与体内受精比较,各蛋白无差异。4.IVM的MⅡ卵母细胞INSIG-SCAP-SREBP的表达变化依据上述表型及血脂结果,血压及血脂变化主要发生于IVM组,因此卵子研究以400个IVM的MⅡ卵母细胞为实验对象,400个体内成熟卵子为对照。(1) mRNA水平：Insig1, Scap, Srebf1基因的表达在IVM卵子上的表达显著高于体内成熟卵子,并有统计学意义。另外,IVM卵子的miR-33及miR-122表达显著低于体内成熟卵子。(2)蛋白水平：INSIG1蛋白及SCAP蛋白在IVM卵子与体内成熟卵子之间,表达差异存在统计学意义。结论：1.IVM可影响子代老年时期的血压。2.IVM及ICSI技术可导致子代老年时期血脂、血胰导素及器官病理改变。3.ART影响子代老年时期血脂代谢可能与INSIG-SCAP-SREBP的表达变化相关。4.ART导致INSIG-SCAP-SREBP的表达变化可能与它们CpG岛的甲基化率改变及相应的微小,RNA的表达改变有关。5.IVM卵子上的INSIG-SCAP-SREBP的表达变化,进一步验证了胚源性疾病学说。更多还原

【Abstract】 Part I Effects of IVF on growth and expression of Igf2/H19and their epigenetic mechanisms in the liver and skeletal muscleObjective:To investigate the growth, gene expression of Igf2, H19, Igf2r, miRNA, and methylation status of Igf2/H19in the liver and skeletal muscle by establishing mouse model of IVF, and to illuminate the short-term and long-term effects of IVF and mechanism involved.Materials and methods:1. To establish models of IVF and in vivo fertilization by using C57BL/6J mice. The in vivo group was used as the control group, and the effects of IVF on birth outcome were detected by comparing the pregnancy rate, gestation, birth number, sex ratio, litter size, and death ratio.2. The effects of IVF on development were detected by checking the growth weight from birth to the sex matured stage (10wk), and the specific gravity of organs at3wk,10wk, and1.5yr.3. The effects of IVF on gene expression of Igf2, H19, Igf2r, and miRNA-483were detected at birth,3wk,10wk, and1.5yr by real-time quantitative PCR.4. The methylation status of Igf2/H19DMRs in mouse liver and skeletal muscle was first determined by bisulfite PCR and clone sequencing. Then, the changed CpG sites of Igf2/H19DMR were verified by pyrosequencing. 5. The IGF2protein expression in the liver and skeletal muscle of IVF mice was detected at birth,3wk,10wk, and1.5yr by western blotting.Result (s):1. A total of51IVF and51in vivo mice were delivered. There were no significant differences between the IVF and in vivo groups in the gestation, litter size, birth death ratio, sex ratio, and pregnancy rate.2. At birth, the body weight of IVF mice was significantly higher than that of in vivo mice. At2wk and3wk, the body weight of IVF mice was significantly lower than that of in vivo mice. After3wk, the difference in body weight between the two groups disappeared. There were no significant differences in the weight of organs at all the time points between the two groups. However, the weight of spleen in IVF mice was significantly lower than that of in vivo mice at10wk.3. Effects of IVF on the expression levels of Igf2, H19, Igf2r, and miRNA-483(1) Expression levels of Igf2, H19, Igf2r, and miRNA-483in liverAt birth and3wk, there were significant differences in gene expression of Igf2, H19and miRNA-483between IVF mice and in vivo mice. At10wk, the difference in gene expression between IVF and in vivo mice disappeared. However, when the age reached to1.5yr, the significant differences in gene expression of Igf2, H19and miRNA-483were detected again between the two group mice. In addition, there were no differences in gene expression of Igf2r between the two groups from birth to old age.(2) Expression levels of Igf2, H19, Igf2r, and miRNA-483in skeletal muscleAt3wk, there were significant differences in gene expression of Igf2, H19, and miRNA-483. At10wk, no significant differences in gene expression of Igf2, H19and miRNA-483were found between two groups. However, when the age reached to1.5yr, the significant differences in gene expression of Igf2, H19and miRNA-483were detected again between the two group mice. At3wk and10wk, there were no differences in gene expression of Igf2r between the two groups. However, at1.5yr, gene expression of Igf2r was significantly lower in IVF mice than in control mice.4. Effects of IVF on the DNA methylation status of Igf2/H19and Igf2r DMRs Given the disrupted expression levels of Igf2/H19, the DNA methylation rate of the H19DMR (21lbp,12CpG sites) and Igf2DMR2(238bp,13CpG sites) were first calculated by cloning and sequencing. Among all the CpG sites of H19DMR, the CpG6, CpG7, and CpG8showed differences between the two groups. Also, the CpG4and CpG5sites of Igf2DMR2showed differences. Thus, the DNA methylation rate of the H19DMR (CpG6-8sites) and Igf2DMR2(CpG1-5sites) were chosen to verify by pyrosequencing. Further, we added Igf2r DMR2(4CpG sites) to analyze the DNA methylation status.(1) DNA methylation status of Igf2/H19and Igf2r DMRs in liver1)At birth, IVF mice showed significant difference in the methylation rate of H19DMR. At3wk and10wk, there was no significant difference in the methylation rate of H19DMR between the two groups. However, when the age reached to1.5yr, the significant differences in the methylation rate of H19DMR was detected again between the two group mice.2) At birth and3wk, there was significant difference in the methylation rate of Igf2DMR2between IVF and control mice. At10wk, there was no significant difference in the methylation rate of Igf2DMR2between the two groups. However, when the age reached to1.5yr, the significant differences in the methylation rate of H19DMR was detected again between the two group mice.3) There was no difference in the methlyation rate of Igf2r DMR2between the two groups of mice from birth to old age.(2) DNA methylation status of Igf2/Hl9DMRs and Igf2r in skeletal muscle1) At3wk, IVF mice showed significantly lower methylation rate of H19DMR when compared with in vivo mice. There was no significant difference in H19DMR between the two groups at10wk and old age.2) At3wk, there was significant difference in the methylation rate of Igf2DMR2between IVF mice and in vivo mice. The difference of methylation rate of Igf2DMR2in IVF mice disappeared at10wk. However, when the age reached to1.5yr, the significant difference in the methylation rate of Igf2DMR2was detected again between the two group mice. 3) At3wk and10wk, the methlyation rate of Igf2r DMR2of IVF mice was similar to that of in vivo mice. However, at old age, the methylation rate of Igf2r DMR was significantly higher in IVF mice than in control mice.5. Effects of IVF on the IGF protein expression in the liver and skeletal muscleAt3wk, both in the liver and skeletal muscle, the expression of IGF2protein was significantly lower in IVF mice than in control mice. However, there was no statistically significant difference in IGF2protein expression between IVF and in vivo groups at other stages.Conclusion (s):1. IVF could affect the early body growth, and the aberrant growth may be restored with age.2. The effects of IVF on gene expression of Igf2, H19and miRNA-483in the liver and skeletal muscle not only in newborn mice but also in elder mice.3. The aberrant growth that was noted along with Igf2/H19differences in IVF offspring may be associated with epigenetic mechanisms such as DNA methylation and miR-483. Part II Effects of ART on blood lipid metabolism in elder mice and mechanism involvedObjective:To investigate the effects of ART on blood lipid metabolism and mechanism involved, to determine the risk of ART induced adult disease, and to illuminate the main etiological factor and etiological time by ART.Materials and methods:1. Investigation on the phenotype and blood lipid metabolism of elder mice(1) Mice of the C57BL/6J were used as oocyte and sperm donors. Superovulated mouse oocytes were fertilized in vivo constituted the control group. The ART groups were established containing IVF, ICSI, and IVM conceived mice. In total,16controls,12IVF-conceived,12ICSI-conceived,10IVM-conceived mice were examined at1.5years of age.(2) The spatial learning and memory capability were detected by using the water maze and fear condition tests at1.5yr.(3) The blood pressure (BP) and heart ratio (HR) were analyzed by using a programmed sphygmomanometer.(4) Intraperitoneal glucose tolerance testing (IPGTT) was used to test the fasting glucose level and glucose tolerance.(5) Plasma chemistry was detected by Clinical Chemistry Analysers. Serum insulin level was determined using Rat/Mouse Insulin ELISA kit.(6) At1.5yr, some viscera were excised and weighed. In addition, histopathology analyses were done. Samples were fixed in4%paraform and embedded in paraffin wax. Sections were stained with hematoxilyn-eosin (HE) stain.2. Investigation on the involved mechanism Based on the results of blood lipid metabolism and pathologic changes in organs, the regulative pathway of INSIG-SCAP-SREBP and miRNA were analyzed in the liver and lung of elder mice conceived by ART and in the IVM oocytes.(1) mRNA expression of INSIG-SCAP-SREBP in elder miceGene expressions of Insigl, Insig2, Scap, Srebfl, Srebf2, miR-33and miR-122were analyzed by real-time quantitative PCR.(2) DNA methylation status of INSIG-SCAP-SREBP in elder miceThe methylation status of CpG inslands of Insig-Scap-Srebp was determined by BSP and pyrosequencing.(3) Protein expression of INSIG-SCAP-SREBP in elder miceThe protein expressions of INSIG-SCAP-SREBP were detected by western blotting or immunohistochemistry.(4) Expression of INSIG-SCAP-SREBP in the MⅡ oocytesExpressions of INSIG-SCAP-SREBP in the MⅡ oocytes were analyzed by real-time quantitative PCR and fluorescence immunocytochemistry.Result (s):1. Effects of ART on the phenotype and blood lipid metabolism(1) No differences were detected in the learning and memory ability including incubation period, the times of crossing, and freezing percent between ART and in vivo groups.(2) In the IVM treatment group, the BP and HR were significantly higher than other three groups at old age. No differences of BP and HR were found in ICSI and IVF groups as compared with the control group.(3) As the IPGTT shown, only30min after glucose injection, the serum glucose level in the ICSI group was significantly lower than that of in vivo group.(4) The results showed that when compared with the in vivo group, IVM or ICSI-conceived elder mice revealed significant differences in total cholesterol, LDL-C, HDL-C, apolipoprotein-A1, AST/ALT, and CRP. In addition, higher serum insulin level was found in IVM conceived mice.(5) In the ICSI and IVM groups, the heart and kidney weight were significantly higher than IVF group and in vivo group. Moreover, in the IVM group, the pancreas and liver weight were significantly higher than other three groups.(6) As HE stains shown, fatty degeneration of a kidney in IVM mice was observed. In addition, the pathological changes were found in the lung of IVF and ICSI conceived mice.2. Expression levels of INSIG-SC AP-SREBP in the liver of aged ART miceGiven the aberrant blood lipid metabolism in the IVM and ICSI aged mice, ART mice containing IVF, ICSI, and IVM were chosen to analyze. The in vivo fertilization constituted the control group.(1) Gene expressions of Insigl, Scap, Srebfl, and Srebf2were significantly higher in IVM and ICSI groups than in IVF and control groups (2-4folds, P<0.01). In addition, gene expressions of miR-33and miR-122were significantly lower in IVM and ICSI groups when compared with IVF and control groups.(2)Both IVM and ICSI groups showed significant differences in the DNA methylation rate of Insigl, Scap, Srebfl, and Srebf2.(3) The protein expression of SCAP was significantly higher in IVM and ICSI groups as compared with IVF and in vivo groups.3. Expression levels of INSIG-SCAP-SREBP in the lung of aged ART miceGiven the pathologic changes in the lung of IVF and ICSI aged mice, ART mice containing IVF and ICSI were chosen to analyze. The in vivo fertilization constituted the control group.(1) Gene expressions of Insigl, Insig2, Scap, Srebfl, and Srebf2were significantly lower in IVF and ICSI groups than in control group.(2) Both IVF and ICSI groups showed significant differences in the DNA methylation rate of Insigl, Scap, Srebf1, and Srebf2. Moreover, there are significant differences in the DNA methylation rate of Insig-Scap-Srebf between abnormal lung and normal lung. (3) No differences in protein expressions of INSIG-SCAP-SREBP were detected between ART groups and control group.4. Expression levels of INSIG-SCAP-SREBP in the MⅡ oocytesGiven the abnormal blood pressure and blood fat in the IVM aged mice, the IVM oocytes (n=400) were chosen as the experimental group, and in vivo maturation oocytes (n=400) were chosen as the control group.(1) Gene expressions of Insigl, Scap, and Srebf1were significantly higher in IVM oocytes than in control oocytes. In addition, gene expressions of miR-33and miR-122were significantly lower in IVM oocytes than in control oocytes.(2) There are significant differences in protein expressions of INSIG1and SCAP between IVM oocytes and in vivo oocytes.Conclusion (s):1. IVM may induce higher blood pressure.2. Both IVM and ICSI could result in the changes of blood lipid metabolism, serum insulin, and organ morphous structure.3. ART can result in a higher risk of lipid metabolic abnormality in elder mice, which may be associated with alterations in gene expression of Insig-Scap-Srebf.4. The aberrant blood lipid metabolism that was noted along with Insig-Scap-Srebf differences in ART offspring may be linked with epigenetic mechanisms such as DNA methylation and miRNA.5. The effects of IVM on the expression of INSIG-SCAP-SREBP in oocytes, which verified the theory of embryo-fetal diseases.更多还原