卵胞浆内单精子注射（ICSI）技术对小鼠肾脏疾病相关印记基因表达及甲基化状态的影响

发布时间：2018-06-06 01:19

本文选题：卵胞浆内单精子注射技术 + 肾脏　；参考：《浙江大学》2014年博士论文

【摘要】：自1978年人类首例体外受精(in vitro fertilization and embryo transfer, IVF)婴儿出生以来,以IVF和卵胞浆内单精子注射(intracytoplasmic sperm injection, ICSI)为主流技术的辅助生殖技术(assisted reproductive techonologies, ART)已经成为越来越多不孕家庭选择的不孕症治疗技术。ART子代的健康状况,尤其是ICSI子代的健康已经成为ART技术安全性的研究热点之一。 ICSI技术主要针对男性少、弱、畸精子症患者,这些患者因精子数量过少或形态功能障碍,不能穿透卵母细胞透明带,无法完成精卵融合,因而无法完成受精过程或导致受精率显著下降。ICSI技术不仅包括配子和胚胎的体外培养的过程,且其受精过程是通过显微注射系统人为选择精子,并直接将其注入卵细胞胞浆内。人为选择精子意味着避开了卵子对精子的自然选择机制,同时,注射过程可对卵子形成轻微的机械性损伤,并带入一些异源性物质,如聚乙烯吡咯酮等,这些操作可能对配子的受精和后续胚胎的分化和发育产生影响。从ICSI技术开展以来,陆续有大规模流行病学研究报导ICSI子代存在出生缺陷的风险较正常儿童高,但子代出现生长发育异常的机制目前尚未明确。研究发现哺乳动物的发育先后经历两次全基因组DNA甲基化重新编程：第一次在原始生殖细胞发育期全基因组范围内去甲基化,而后在配子成熟过程中重新获得甲基化；第二次发生在胚胎植入前基因组DNA再次发生去甲基化,印记基因则逃避了去甲基化过程,其甲基化水平维持不变。ICSI技术不仅涉及配子短期体外培养过程,而且在卵子MⅡ期干扰了其正常受精过程并涉及植入前胚胎发育过程,因此可能对印记基因甲基化状态的获得和维持产生影响。对ICSI子代研究发现差异甲基化区(differentially methylated region, DMR)的甲基化修饰改变可能是导致其出生缺陷的原因之一。已有的回顾性流行病学调查表明ICSI子代存在泌尿道畸形发生率升高的趋势,同时,已知一些印记基因,如MEST、H19、IGF2、PEG3、SNRPN、CDKNLC、 SOCS-3、SFRP1、RASAL1等,与肾脏发育缺陷、肾脏纤维化及肾脏肿瘤性疾病的发生关系密切。ICSI技术是否可通过干扰肾脏基因印记而影响ICSI子代肾脏疾病的发生是有待解答的ART安全性问题之一本研究建立ICSI小鼠模型(实验组)和2细胞胚胎移植小鼠模型(对照组),比较分析ICSI出生小鼠与对照小鼠子代父源印记基因H19以及母源印记基因Igf2、Snrpn、Mest和Peg3的表达差异,检测了相关印记基因DMR的甲基化状态,分析了ICSI技术对子代肾脏疾病相关印记基因表达的影响,探究了相关印记基因表达异常可能存在的表观遗传学机制,为ICSI技术的安全性评估理论依据。第一部分ICSI技术对小鼠肾脏疾病相关印记基因H19、Igf2、Mest、Peg3和Snrpn的表达的影响目的：建立ICSI小鼠模型(实验组)和2细胞胚胎移植小鼠模型(对照组),研究ICSI子代小鼠肾脏中肾脏疾病相关印记基因H19、Igf2、Mest、Peg3和Snrpn的表达与2细胞胚胎移植组子代是否存在差异,阐明ICSI技术对目的印记基因表达的影响。方法：利用C57BL/6J小鼠,建立ICSI小鼠模型和2细胞胚胎移植小鼠模型,出生小鼠饲养至成年期(10周)和老年期(1.5年),收集两组小鼠的肾脏组织,两个时期各自收集ICSI小鼠肾脏8例和2细胞胚胎移植小鼠肾脏10例,比较两组小鼠及其肾脏的重量,并采用逆转录实时荧光定量PCR检测肾脏中目的基因mRNA的表达水平。结果：在成年子代中,ICSI小鼠肾脏中H19、Mest、Peg3和Snrpn的表达水平较对照组均显著上调。而Igf2的表达水平无显著差异。而在老年子代中,ICSI组snrpn较对照组呈显著下调,而其余4个基因与对照组相比无显著差异。结论：ICSI子代肾脏中存在印记基因H19、Mest、Peg3和Snrpn的表达异常,可能影响ICSI子代肾脏疾病的发生。第二部分ICSI技术对小鼠肾脏中H19DMR、MestDMR、 Peg3DMR和SnrpnDMR的甲基化水平的影响目的：研究ICSI子代肾脏中H19DMR、MestDMR、Peg3DMR和SnrpnDMR的甲基化状态,比较其与对照组之间的差异,并探索其与基因表达间的关系。方法：选择成年期和老年期收集的小鼠肾脏组织,两个时期各自收集ICSI小鼠肾脏8例和2细胞胚胎移植小鼠肾脏10例,采用亚硫酸盐处理后克隆测序(bisulfite sequencing, BSP)法检测ICSI组与对照组H19DMR和SnrpnDMR的DNA甲基化状态,并采用焦磷酸盐测序法对其中部分CpG位点进行验证,同时采用焦磷酸盐测序法对MestDMR和Peg3DMR区DNA甲基化状态进行检测。结果：成年ICSI子代肾脏H19DMR、MestDMR、Peg3DMR甲基化水平较对照组均显著降低,与其调控的基因的表达量较对照组升高均相符；成年ICSI子代肾脏SnrpnDMR甲基化水平与对照组呈现降低趋势,但未达到统计学显著性差异；老年ICSI子代肾脏SnrpnDMR甲基化水平呈显著升高,与Snrpn基因的相对表达量降低相符。结论：成年ICSI子代肾脏中H19、Mest、Peg3基因mRNA表达水平上调可能与基因DMR的甲基化水平改变有关,老年ICSI子代肾脏中Snrpn基因的相对表达量降低也可能与其DMR高甲基化水平相关。ICSI技术可能干扰了早期配子受精及胚胎植入前的甲基化状态。随着年龄增长,ICSI小鼠SnrpnDMR甲基化水平可能重新编程。
[Abstract]:Since the birth of the first human in vitro fertilization and embryo transfer (IVF) in 1978, an increasing number of sterile families have been chosen for the mainstream technology of single sperm injection in IVF and ooplasm (intracytoplasmic sperm injection, ICSI). The health status of.ART offspring, especially the health of ICSI offspring, has become one of the research hotspots of ART technology security.
ICSI technology is mainly aimed at less male, weak and abnormal spermatospermia. These patients are unable to penetrate the oocyte zona pellucid and fail to complete the ovum fusion because of the oligozoospermia or dysfunctional spermatozoa, which can not complete the process of fertilization or lead to a significant decrease in the rate of fertilization, which includes the culture of the gametes and embryos in vitro, and the process of.ICSI is not only included in the culture of gametes and embryos. The process of fertilization is to artificially select sperm by the microinjection system and directly inject it into the cytoplasm of the egg cells. Artificial selection of sperm means avoiding the natural selection mechanism of the egg to the sperm. At the same time, the injection process can form a slight mechanical damage to the egg and bring into some heterogenous substances, such as polyvinylpyrrolidone, and so on. It may affect the fertilization of gametes and the differentiation and development of subsequent embryos.
Since the development of ICSI technology, there has been a large-scale epidemiological study that the risk of birth defects in the ICSI progeny is higher than that of normal children. However, the mechanism of the growth and development of the offspring is not yet clear. The study found that the development of mammalian development has undergone two complete genome DNA methylation reprogramming: the first reproduction in primitive reproduction. Methylation in the whole genome range of the cell development period and then re methylation during the gamete maturation; the second occurs before the implantation of the genomic DNA, and the imprinting gene evade the demethylation process. The methylation level maintaining.ICSI technology not only involves the short in vitro culture of gametes, but also the methylation level remains unchanged. And the M II phase interferes with the normal fertilization process and involves the development of the preimplantation embryo, which may affect the acquisition and maintenance of the methylation status of the imprinted genes. The study of the ICSI progeny study found that the methylation modification of the differential methylation zone (differentially methylated region, DMR) may lead to its birth defects. One of the reasons.
A retrospective epidemiological survey has shown that the incidence of urinary tract malformation in the ICSI progeny is higher. At the same time, some of the known imprinting genes, such as MEST, H19, IGF2, PEG3, SNRPN, CDKNLC, SOCS-3, SFRP1, RASAL1, are closely related to kidney development, renal fibrosis and renal tumor. It is one of the ART safety problems to be answered to interfere with renal gene imprinting and to influence the occurrence of renal diseases in ICSI progeny.
In this study, the ICSI mice model (experimental group) and 2 cell embryo transfer mice model (control group) were established. The differences in the expression of the parent imprinted gene H19 and the parent imprinted gene Igf2, Snrpn, Mest and Peg3 in the ICSI born mice and the control mice were compared and analyzed. The methylation status of the associated gene DMR was detected, and the ICSI technique was analyzed for the progeny of the offspring. The influence of the expression of the imprinted gene on renal disease related to the epigenetic mechanism that may exist in the related imprinting gene expression, which is the theoretical basis for the safety assessment of ICSI technology.
Part 1 the effect of ICSI technology on the expression of H19, Igf2, Mest, Peg3 and Snrpn genes related to kidney diseases in mice.
Objective: to establish ICSI mice model (experimental group) and 2 cell embryo transfer mice model (control group), to investigate whether the expression of renal disease related imprint gene H19, Igf2, Mest, Peg3 and Snrpn in the kidney of ICSI offspring mice is different from that of the 2 cell embryo transfer group, and clarifying the effect of ICSI technology on the expression of the target gene expression.
Methods: C57BL/6J mice were used to establish a ICSI mouse model and a 2 cell embryo transfer mouse model. The mice were bred to adult (10 weeks) and aged (1.5 years), and two groups of kidney tissues were collected. 8 cases of ICSI mice and 10 kidneys of 2 cell embryo transfer mice were collected in two periods, and the weight of the two mice and their kidneys were compared. Quantitative reverse transcription real time fluorescence quantitative PCR was used to detect the expression level of target gene mRNA in kidney.
Results: in the adult offspring, the expression level of H19, Mest, Peg3 and Snrpn in the kidney of ICSI mice was significantly higher than that in the control group, but there was no significant difference in the expression level of Igf2. In the older generation, the SNRPN of the ICSI group was significantly lower than the control group, but the other 4 genes were not significantly different from the control group.
Conclusion: abnormal expression of imprinted genes H19, Mest, Peg3 and Snrpn in ICSI progeny kidneys may affect the occurrence of renal diseases in ICSI offspring.
The second part is the effect of ICSI technology on the methylation level of H19DMR, MestDMR, Peg3DMR and SnrpnDMR in mouse kidneys.
Objective: To study the methylation status of H19DMR, MestDMR, Peg3DMR and SnrpnDMR in the kidneys of ICSI offspring, and to compare the difference between the control group and the control group, and to explore the relationship between them and the gene expression.
Methods: the mice kidney tissues collected in adult and old age were selected. 8 cases of ICSI mice kidney and 10 mice with 2 cell embryos were collected at two periods. The methylation of H19DMR and SnrpnDMR in ICSI group and control group was detected by bisulfite sequencing, BSP method, and pyrophosphoric acid was used. Salt sequencing method was used to verify some of the CpG loci, and pyrophosphate sequencing was used to detect the DNA methylation status in MestDMR and Peg3DMR regions.
Results: the levels of H19DMR, MestDMR, and Peg3DMR methylation in the adult ICSI offspring were significantly lower than those in the control group, and the expression of the gene was in accordance with the control group. The SnrpnDMR methylation level in the kidney of the adult ICSI progeny was lower than that in the control group, but there was no significant difference between the control and the control group, and the renal Snr of the elderly ICSI progeny was Snr. The level of pnDMR methylation was significantly increased, which was in line with the relative reduction of Snrpn gene expression.
Conclusion: the up-regulated level of mRNA expression of H19, Mest, Peg3 gene in the adult ICSI progeny may be related to the change of DMR methylation level. The decrease of the relative expression of Snrpn gene in the kidney of the elderly ICSI progeny may also be associated with the DMR hypermethylation level.ICSI technology may interfere with the early gamete fertilization and the methyl methylation before the implantation of the embryo. The SnrpnDMR methylation level of ICSI mice may be reprogrammed with age.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.8

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