本文選題：卵胞漿內(nèi)單精子注射技術(shù) + 腎臟��； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：自1978年人類首例體外受精(in vitro fertilization and embryo transfer, IVF)嬰兒出生以來,以IVF和卵胞漿內(nèi)單精子注射(intracytoplasmic sperm injection, ICSI)為主流技術(shù)的輔助生殖技術(shù)(assisted reproductive techonologies, ART)已經(jīng)成為越來越多不孕家庭選擇的不孕癥治療技術(shù)。ART子代的健康狀況,尤其是ICSI子代的健康已經(jīng)成為ART技術(shù)安全性的研究熱點(diǎn)之一。 ICSI技術(shù)主要針對(duì)男性少、弱、畸精子癥患者,這些患者因精子數(shù)量過少或形態(tài)功能障礙,不能穿透卵母細(xì)胞透明帶,無法完成精卵融合,因而無法完成受精過程或?qū)е率芫曙@著下降。ICSI技術(shù)不僅包括配子和胚胎的體外培養(yǎng)的過程,且其受精過程是通過顯微注射系統(tǒng)人為選擇精子,并直接將其注入卵細(xì)胞胞漿內(nèi)。人為選擇精子意味著避開了卵子對(duì)精子的自然選擇機(jī)制,同時(shí),注射過程可對(duì)卵子形成輕微的機(jī)械性損傷,并帶入一些異源性物質(zhì),如聚乙烯吡咯酮等,這些操作可能對(duì)配子的受精和后續(xù)胚胎的分化和發(fā)育產(chǎn)生影響。 從ICSI技術(shù)開展以來,陸續(xù)有大規(guī)模流行病學(xué)研究報(bào)導(dǎo)ICSI子代存在出生缺陷的風(fēng)險(xiǎn)較正常兒童高,但子代出現(xiàn)生長(zhǎng)發(fā)育異常的機(jī)制目前尚未明確。研究發(fā)現(xiàn)哺乳動(dòng)物的發(fā)育先后經(jīng)歷兩次全基因組DNA甲基化重新編程：第一次在原始生殖細(xì)胞發(fā)育期全基因組范圍內(nèi)去甲基化,而后在配子成熟過程中重新獲得甲基化；第二次發(fā)生在胚胎植入前基因組DNA再次發(fā)生去甲基化,印記基因則逃避了去甲基化過程,其甲基化水平維持不變。ICSI技術(shù)不僅涉及配子短期體外培養(yǎng)過程,而且在卵子MⅡ期干擾了其正常受精過程并涉及植入前胚胎發(fā)育過程,因此可能對(duì)印記基因甲基化狀態(tài)的獲得和維持產(chǎn)生影響。對(duì)ICSI子代研究發(fā)現(xiàn)差異甲基化區(qū)(differentially methylated region, DMR)的甲基化修飾改變可能是導(dǎo)致其出生缺陷的原因之一。 已有的回顧性流行病學(xué)調(diào)查表明ICSI子代存在泌尿道畸形發(fā)生率升高的趨勢(shì),同時(shí),已知一些印記基因,如MEST、H19、IGF2、PEG3、SNRPN、CDKNLC、 SOCS-3、SFRP1、RASAL1等,與腎臟發(fā)育缺陷、腎臟纖維化及腎臟腫瘤性疾病的發(fā)生關(guān)系密切。ICSI技術(shù)是否可通過干擾腎臟基因印記而影響ICSI子代腎臟疾病的發(fā)生是有待解答的ART安全性問題之一 本研究建立ICSI小鼠模型(實(shí)驗(yàn)組)和2細(xì)胞胚胎移植小鼠模型(對(duì)照組),比較分析ICSI出生小鼠與對(duì)照小鼠子代父源印記基因H19以及母源印記基因Igf2、Snrpn、Mest和Peg3的表達(dá)差異,檢測(cè)了相關(guān)印記基因DMR的甲基化狀態(tài),分析了ICSI技術(shù)對(duì)子代腎臟疾病相關(guān)印記基因表達(dá)的影響,探究了相關(guān)印記基因表達(dá)異�？赡艽嬖诘谋碛^遺傳學(xué)機(jī)制,為ICSI技術(shù)的安全性評(píng)估理論依據(jù)。 第一部分ICSI技術(shù)對(duì)小鼠腎臟疾病相關(guān)印記基因H19、Igf2、Mest、Peg3和Snrpn的表達(dá)的影響 目的：建立ICSI小鼠模型(實(shí)驗(yàn)組)和2細(xì)胞胚胎移植小鼠模型(對(duì)照組),研究ICSI子代小鼠腎臟中腎臟疾病相關(guān)印記基因H19、Igf2、Mest、Peg3和Snrpn的表達(dá)與2細(xì)胞胚胎移植組子代是否存在差異,闡明ICSI技術(shù)對(duì)目的印記基因表達(dá)的影響。 方法：利用C57BL/6J小鼠,建立ICSI小鼠模型和2細(xì)胞胚胎移植小鼠模型,出生小鼠飼養(yǎng)至成年期(10周)和老年期(1.5年),收集兩組小鼠的腎臟組織,兩個(gè)時(shí)期各自收集ICSI小鼠腎臟8例和2細(xì)胞胚胎移植小鼠腎臟10例,比較兩組小鼠及其腎臟的重量,并采用逆轉(zhuǎn)錄實(shí)時(shí)熒光定量PCR檢測(cè)腎臟中目的基因mRNA的表達(dá)水平。 結(jié)果：在成年子代中,ICSI小鼠腎臟中H19、Mest、Peg3和Snrpn的表達(dá)水平較對(duì)照組均顯著上調(diào)。而Igf2的表達(dá)水平無顯著差異。而在老年子代中,ICSI組snrpn較對(duì)照組呈顯著下調(diào),而其余4個(gè)基因與對(duì)照組相比無顯著差異。 結(jié)論：ICSI子代腎臟中存在印記基因H19、Mest、Peg3和Snrpn的表達(dá)異常,可能影響ICSI子代腎臟疾病的發(fā)生。 第二部分ICSI技術(shù)對(duì)小鼠腎臟中H19DMR、MestDMR、 Peg3DMR和SnrpnDMR的甲基化水平的影響 目的：研究ICSI子代腎臟中H19DMR、MestDMR、Peg3DMR和SnrpnDMR的甲基化狀態(tài),比較其與對(duì)照組之間的差異,并探索其與基因表達(dá)間的關(guān)系。 方法：選擇成年期和老年期收集的小鼠腎臟組織,兩個(gè)時(shí)期各自收集ICSI小鼠腎臟8例和2細(xì)胞胚胎移植小鼠腎臟10例,采用亞硫酸鹽處理后克隆測(cè)序(bisulfite sequencing, BSP)法檢測(cè)ICSI組與對(duì)照組H19DMR和SnrpnDMR的DNA甲基化狀態(tài),并采用焦磷酸鹽測(cè)序法對(duì)其中部分CpG位點(diǎn)進(jìn)行驗(yàn)證,同時(shí)采用焦磷酸鹽測(cè)序法對(duì)MestDMR和Peg3DMR區(qū)DNA甲基化狀態(tài)進(jìn)行檢測(cè)。 結(jié)果：成年ICSI子代腎臟H19DMR、MestDMR、Peg3DMR甲基化水平較對(duì)照組均顯著降低,與其調(diào)控的基因的表達(dá)量較對(duì)照組升高均相符；成年ICSI子代腎臟SnrpnDMR甲基化水平與對(duì)照組呈現(xiàn)降低趨勢(shì),但未達(dá)到統(tǒng)計(jì)學(xué)顯著性差異；老年ICSI子代腎臟SnrpnDMR甲基化水平呈顯著升高,與Snrpn基因的相對(duì)表達(dá)量降低相符。 結(jié)論：成年ICSI子代腎臟中H19、Mest、Peg3基因mRNA表達(dá)水平上調(diào)可能與基因DMR的甲基化水平改變有關(guān),老年ICSI子代腎臟中Snrpn基因的相對(duì)表達(dá)量降低也可能與其DMR高甲基化水平相關(guān)。ICSI技術(shù)可能干擾了早期配子受精及胚胎植入前的甲基化狀態(tài)。隨著年齡增長(zhǎng),ICSI小鼠SnrpnDMR甲基化水平可能重新編程。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.8

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相關(guān)期刊論文 前2條

1 ;Expression of SOCS-3 and Its Methylation in Renal Cancer[J];Chemical Research in Chinese Universities;2011年03期

2 Yue-hong LU;Ning WANG;Fan JIN;;Long-term follow-up of children conceived through assisted reproductive technology[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2013年05期

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本文編號(hào)：1984335