【作者】 邵伟娟；

【导师】 赵茹茜；

【作者基本信息】 南京农业大学 ， 动物学， 2007， 博士

【摘要】 本研究利用糖尿病模型小鼠和胰岛素处理体外培养的2细胞胚胎至囊胚期,研究早期发育环境中的胰岛素对F1和F2代生长和胰岛功能的代谢程序化影响;通过检测印迹基因Igf2和H19在早期胚胎的表达和印迹控制区DNA甲基化的变化;采用基因表达谱芯片筛选相关的差异表达基因,以探讨胰岛素胎儿程序化作用的可能机理。1.胰岛素对小鼠早期胚胎发育的程序化影响1.1不同剂量STZ诱导糖尿病模型小鼠通过一次性腹腔注射150 mg/kg b.w.和200 mg/kg b.w.的链脲菌素(STZ)以制作小鼠糖尿病模型;采用葡萄糖测定试纸和尿液分析试纸条联合检测模型小鼠血糖和尿糖的变化。注射STZ后第4周测定小鼠血清葡萄糖和胰岛素浓度,光镜观察胰岛的组织学改变,并使模型小鼠与正常雄鼠交配,观察交配和妊娠情况。结果表明:对照组血糖在观测期基本无变化,模型组血糖水平逐渐增加,三周后稳定。高剂量组血清葡萄糖浓度明显升高(P＜0.001),血清胰岛素水平极显著下降(P＜0.001);胰岛形态破坏严重;低剂量组小鼠血清葡萄糖分升高组和正常组,但血清胰岛素浓度均明显降低(P＜0.01),胰岛内可见炎性细胞浸润。血糖升高组小鼠交配能力下降,交配率仅为46.9%,流产率为73.9%,仔鼠出生死亡率为20%。结论:不同剂量的STZ均能诱发糖尿病,且影响雌鼠的交配能力和胚胎的生长发育。1.2母体低胰岛素、高血糖环境对子代生长发育和胰岛功能的程序化影响STZ诱导的糖尿病模型小鼠与正常雄鼠交配产下F1代,F1代雌鼠成年后,与正常雄鼠远交产下F2代,观察母体低胰岛素、高血糖环境对子代(F1和F2)生长发育和胰岛功能的程序化影响。结果显示:试验组F1代畸形率为9%,仔鼠出生重下降26.5%(P＜0.01)。F1代试验组血清葡萄糖浓度比对照组升高24.7%(P＜0.05),血清胰岛素浓度比对照组下降26%(P＜0.05)。F2代试验组血清葡萄糖浓度与对照组没有差异,但血清胰岛素浓度比对照组下降14.3%(P＜0.05)。F1代、F2代成年鼠胰岛素耐量损伤,产生胰岛素抵抗,胰岛凋亡细胞比例升高。提示:糖尿病母体环境影响子代胚胎发育和成年后产生胰岛素抵抗,这种影响可以传递至F2代。1.3植入前胚胎胰岛素处理对子代生长发育和胰岛功能的程序化影响将小鼠2细胞胚胎培养于KSOM+0.25μg/ml Insulin的培养液中,发育至囊胚后转移到假孕母鼠子宫。产下的雌鼠成年后(6周龄),与正常雄鼠远交产下F1代,观察暴露于高胰岛素环境的早期胚胎及其子代生长发育和胰岛功能的程序化影响。结果显示:试验组囊胚发育率升高16.4%(P＜0.05),仔鼠出生重升高17.8%(P＜0.05)。试验组成年小鼠血清葡萄糖和胰岛素浓度与对照组均无明显差异,但胰腺凋亡细胞明显增多,并显示口服葡萄糖和胰岛素耐量损伤;F1代试验组血清胰岛素浓度比对照组下降14.3%(P＜0.05),胰岛素耐量损伤,产生胰岛素抵抗。提示:胰岛素能促进早期胚胎生长,但会造成成年以及F1代小鼠胰岛素抵抗。2.胰岛素胎儿程序化作用的机理2.1母体低胰岛素、高血糖环境对早期胚胎印迹基因Igf2和H19表达和甲基化的影响取糖尿病模型母鼠14天胎龄的胚胎,提取全胚胎的总RNA和DNA,检测印迹基因Igf2和H19的表达和印迹控制区(ICR)的甲基化状态。结果显示:试验组14天胚胎中Igf2 mRNA表达的相对丰度比对照组下降15.4%(P＜0.05),Real-time RT-PCR显示Igf2mRNA表达是对照组的0.65倍(P＜0.05),而H19 mRNA表达与对照组无差异。试验组Igf/H19印迹控制区的CpG甲基化率比对照组升高19.1%(P＞0.05)。提示:母体低胰岛素、高血糖环境影响早期胎儿印迹基因Igf2/H19印迹控制区CpG甲基化率,引起Igf2 mRNA表达的改变。2.2植入前胚胎胰岛素处理对小鼠胚胎印迹基因Igf2/H19表达和甲基化的影响将小鼠2细胞胚胎培养于KSOM+0.25μg/ml Insulin的培养基内,发育至桑椹胚和囊胚,囊胚转移到假孕母鼠子宫。提取桑椹胚、囊胚和14天胚胎的总RNA和总DNA,检测印迹基因Igf2和H19的表达和印迹控制区(ICR)的甲基化状态。Real-timeRT-PCR分析表明,试验组桑椹胚Igf2 mRNA表达是对照组的4.7倍,H19表达是对照组的5.7倍;试验组囊胚Igf2 mRNA表达是对照组的1.8倍,H19表达量是对照组的2.3倍;14天胚胎,试验组Igf2和H19 mRNA表达均是对照组的1.5倍(P＜0.05)。BSP测序法分析Igf2/H19印迹控制区的甲基化水平发现,试验组桑椹胚、囊胚和14天胚胎甲基化率分别为7.3%,32.3%和46%,分别比对照组下降86.4%,35.4%和19.2%(P＜0.01)。提示:植入前胚胎暴露于胰岛素可降低Igf2/H19印迹控制区DNA甲基化的水平,从而使Igf2和H19基因表达升高。3.基因表达谱芯片筛选与胎儿程序化作用相关的差异表达基因取糖尿病模型小鼠和对照组小鼠14胎龄的胚胎,提取胚胎的总RNA。逆转录成cDNA,将Cy3和Cy5 2种荧光分别标记到试验组和对照组的cDNA上,并与包含24859个基因的表达谱芯片进行杂交及扫描,重复3次实验,采用Agilent扫描仪进行扫描分析,软件读取数据,结果筛选出差异表达基因397个,其中有328个基因在试验组表达量比对照组大2倍,69个基因在试验组表达量比对照组小2倍,其中糖尿病相关基因Rrad(Ras-Related Associated with Diabetes)和Rasl12上调,该基因与Ⅱ型糖尿病胰岛素抵抗有关;Otx2 and Robo3基因下调,其主要调节脑和神经系统发育及脑细胞分化。提示:母体糖尿病环境影响早期胎儿基因表达,进而影响胚胎发育。更多还原

【Abstract】 The objectives of the present study were(1) to reveal whether an altered intrauterine endocrine milieu in diabetic mice model in vivo and preimplantation embryos exposed to insulin in vitro would cause long term alterations in growth and development,as well as insulin secretion and action later in adult life;(2) to demonstrate the possible trans-generational effect by examining the alterations in growth and insulin secretion and function in F1 and F2 offspring,and(3) to elucidate whether this action involves epigenetic modification of imprinted genes via changes in DNA methylation.In addtion, DNA microarray was employed to identify the differentially expressed genes in fetuses from control and diabetic mice.1.Effect of insulin on the metabolic programming of mouse embryonic development1.1 Establishment of diabetic mouse model by streptozotocin injection in different dosesTo establish type 1 diabetes mellitus(T1DM),mice were received intraperitoneal injection of streptozotocin(STZ) at the doses of 150mg/kg b.w.and 200mg/kg b.w.. Blood and urine glucose levels were detected by glucose and urine analyzing papers respectively.Pancreatic histological changes of islets were observed under light microscope.Serum insulin concentration was detected by radioimmunoassay.The female diabetic mice were mated to the normal male mice,the mating ability and fetal mortality were observed.The glucose concentration in the control group did not show alteration across the investigation period,while that of the STZ group was increasing with the time, reaching stabilized level 3 weeks after STZ injection.Significantly increased(P＜0.001) serum glucose concentration associated with significantly decreased(P＜0.001) serum insulin levels were observed in the high STZ dose group,with obviously impaired pancreatic isletβcells.In the low STZ dose group,serum insulin concentration was decreased(P＜0.01),while serum glucose level increased or unaltered.In increased glucose group(Glucose＞18mmol/L),the success mating rate,abortion rate and fetal mortality were 46.9%,73.9%and 20%,respectively.The results indicate that STZ can induce diabetes mellitus,and affect the female success mating rate and fetal development.1.2 Effect of maternal low insulin and high glucose environment on programming of fetal development and pancreatic islet functionSTZ-induced diabetic mice were mated to the normal male mice to produce F1 offspring.The adult female F1 mice were bred to give birth to F2 offspring.The alteration of fetal development and pancreatic islet function were observed to reveal the maternal effect.The F1 offspring from diabetic mice exhibited malformation rate of 9%, and 26.5%lower birth weight compared with the control group(P＜0.01).The serum glucose concentration was 24.7%higher(P＜0.05),while the serum insulin concentration was 26%lower than that of the control(P＜0.05).The F2 offspring did not show significant alterations in serum glucose level,but the concentration of serum insulin was 14.3%lower than that of the control(P＜0.05).Adult offspring of both F1 and F2 from diabetic mice displayed insulin resistance and higher apoptotic ratio in pancreas.The result demonstrate that altered maternal endocrine and metabolic status(low insulin and high glucose) could exert trans-generational effect on growth and development,as well as insulin secretion and function in F1 and F2 offspring.1.3 Exposure of Mouse Preimplantation Embryos to Insulin influences fetus development and pancreatic islet programming2-cell embryos were cultured in either 0 or 0.25μg/ml insulin to the blastocyst stage and then transferred into pseudo-pregnant recipient mice.The newboms were raised until sexual maturity and were bred with normal males to produce F1 offspring.The ratio of blastocyst in insulin-exposed group is 16.4%higher than that of the control(P＜0.05), and the birth weight of insulin-exposed group was 17.8%higher than that of the control group(P＜0.05).The differences of serum glucose and insulin between insulin-exposed and control group were not significant,but the treated group demonstrated higher apoptotic ratio in pancreas and insulin resistance in adulthood.The concentration of serum insulin in F1 of treated group was 14.3%lower than that of the control(P＜0.05) with insulin resistance in adulthood.The results indicate that insulin can stimulate early-embryo development and induce alterations in pancreas function,with lasting consequences of insulin tolerance in the offspring in adult life.2.Mechanisms involved in the effect of early insulin intervention2.1 Alterations in the expression and methylation levels of imprinted genes H19 and Igf2 of fetuses from Streptozotocin-Induced Diabetic mice The mRNA expression and methylation levels of the growth-related imprinted genes, H19 and Igf2 in fetuses of Streptozotocin-Induced Diabetic mice were analyzed.Total RNA and DNA of E14 fetuses were extracted.The relative abundance of Igf2 mRNA in STZ treated group was decreased by 15.4%(P＜0.05).Real-time reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts,which is associated with 19.1%higher methylation ratio of all CpGs in the targeted region of genomic DNA.The results indicate that maternal diabetes would affect fetal development via altering the expression of impriting genes.The modified genomic DNA methylation status of imprinting genes may account for the change of gene expression.2.2 Exposure of Mouse Preimplantation Embryos to Insulin Alters the Methylation Status of Imprinted Genes H19 and Igf22-cell embryos were cultured in either 0 or 0.25μg/ml insulin to the morula and blastocyst stage,and then transferred into pseudo-pregnant recipient mice.The DNA and RNA of morula,blastocyst and E14 fetuses were extracted to reveal the alterations of expression and methylation levels of the growth-related imprinted genes,H19 and Igf2. Real-time reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression of Igf2 and H19 in insulin-exposed morula was 4.9 and 5.7 fold of control counterparts,in insulin-exposed blastocyst,that was 1.8 and 2.3-fold respectively. In the insulin-exposed fetuses,both of the mRNA expression of Igf2 and H19 were 1.5 fold of the control group(P＜0.05).The methylation ratio of all CpGs in the targeted region of genomic DNA increased by 86.4%,35.4%and 19.2%in the insulin-exposed morula,blastocyst and E14 fetuses,respectively compared with the control counterparts (P＜0.05).The results indicate that insulin exposure during the preimplantation stage modifies the genomic DNA methylation status of imprinted genes,alters the expression and affects fetal development.3.Identification of differentially expressed genes in fetuses from control and diabetic mice using DNA microarrayTotal RNA of E14 diabetic fetuses were extracted and cDNA were reverse transcribed.Cy3 and Cy5 fluorescent dyes were labeled to diabetic and control cDNA respectively,and used to hybridize mouse oligo-array containing 24,859 genes.Data were analyzed by using Agilent microarray scanner system.A total of 397 genes were found to be differentially expressed between the fetuses of diabetic and control mice.328 genes showed a more than 2-fold increase in the expression compared with the control, while 69 genes showed a more than 2-fold down regulation.For example,Otx2 and Robo3,proteins that are involved in cell differentiation,nervous system and brain development,are down-regulated in the fetuses of the diabetic mice.Ras112 and Rrad, that are associated with diabetes,are up-regulated in the fetuses of the diabetic mice.The present study indicate diabetes mellitus can induce differentially expressed genes,and affect fetal development.更多还原