【作者】 郑重；

【导师】 雷蕾；

【作者基本信息】 哈尔滨医科大学 ， 人体解剖学与组织胚胎学， 2012， 博士

【摘要】 目的研究供体细胞的rDNA状态对核移植（NT）胚胎内rDNA活性以及rRNA合成和加工的影响。方法通过建立了B6D2F1品系小鼠的胚胎干细胞（embryonic stem cells, ESCs）、颗粒细胞（cumulus cells, CCs）和胎儿成纤维细胞（mouse embryonic fibroblasts, MEFs）细胞株，检测了这三者在活性NORs数目、rDNA甲基化水平和核仁相关基因表达水平上的差异；之后以这三种细胞为供体细胞，用一步法核移植技术构建了相应的核移植（nuclear transfer, NT）胚胎，同时以单精注射（intracytoplasmic sperm injection, ICSI）胚胎作为对照，考察了4种胚胎在原核形成、核仁蛋白分布、活性NORs数目、rDNA甲基化水平以及核仁相关基因表达等方面的差异。结果1)小鼠ICSI胚胎和NT胚胎在精子注射/激活后5h（hours post activation, hpa）开始出现核仁前体（nucleolar precursorbodies, NPBs），NPBs数目逐渐减少，直至8hpa时趋于稳定并一直持续到原核融合（16-17hpa）；2)干细胞核移植（embryonic stem cell nuclear transfer, ESNT）胚胎原核期仅有一个类原核，平均内含7.20个NPBs；颗粒细胞（cumulus cellnuclear transfer, CCNT）、成纤维细胞核移植（mouse embryonic fibroblast nucleartransfer, MEFNT）胚胎、ICSI胚胎和孤雌（parthenogenetic activation, PA）胚胎在原核期都形成2个原核/类原核，平均每个原核/类原核内分别含3.71、4.27、1.58和1.59个NPBs；3)晚2细胞期的ICSI、ESNT和CCNT胚胎在核仁周围开始出现网状结构。到4细胞期时，ICSI和ESNT胚胎的核仁呈现完全的网状结构，而CCNT胚胎核仁的网状结构仍只出现在周边区域；4)0.8μg/ml放线菌素D（actinomycin D,AD）处理颗粒细胞1h能使其失去rRNA转录和加工活性。以AD处理后的颗粒细胞作为供体构建的NT胚胎，其植入前发育能力和未处理组并无显著差异；5) ESCs有最多的活性核仁组织区（nucleolar organizingregions， NORs）数目（7.66）和最低的rDNA甲基化水平（6.76%），而且其UBF(upstream binding factor，上游结合因子)、 FBL(fibrillarin)和B23(nucleophosmin，核仁素)的表达水平显著高于CCs和MEFs；MEFs含最少的活性NORs数目（4.70）和最高的rDNA甲基化水平（22.57%）；CCs的数据居于两者之间（6.45和13.59%）；6)4细胞期时，ESNT胚胎含有与ICSI相当的活性NORs数（7.19vs.7.44），显著高于CCNT（6.68）和MEFNT胚胎（5.77）。MEFNT胚胎的rDNA甲基化水平显著高于其他3组胚胎（15.52%vs.9.39%/ICSIvs.6.36%/ESNT vs.9.67%/CCNT）。7) CCs和MEFs的核进入卵母细胞后，B23和UBF信号在20min后消失；而ESCs的核内B23/UBF信号在1h仍能检测到；8) MEFNT胚胎的植入前发育能力低于ICSI、ESNT和CCNT胚胎；桑葚胚期其FBL和18S rRNA的表达显著低于另外三组胚胎，UBF和47S rRNA的表达显著低于ICSI胚胎。结论本实验阐明了供体细胞本身和核移植胚胎内核仁蛋白的分布情况和变化；揭示了不同分化程度的供体细胞在rDNA的活性上存在一定差异，并且这种差异很大程度决定了不同核移植胚胎中的rDNA活性的差别，进而影响核移植胚胎植入前的发育能力。更多还原

【Abstract】 Objective To study the impact of donor cell’s rDNA status on rDNA activity aswell as rRNA synthesis and processing of nuclear transfer (NT) embryos. MethodsWe firstly established mouse embryonic stem cells (ESCs), cumulus cells (CCs) andembryonic fibroblasts (MEFs) cell lines of B6D2F1strain and examined the activeNORs numbers, rDNA methylation levels and nucleoar related genes expression ofthese three cell lines. Then the three kinds of cells were used as donor cells toconstruct NT embryos with one-step NT technique while intracytoplasmic sperminjection (ICSI) embryos were used as control. We examined the pronuclei formation,nucleolar protein distribution, active NORs numbers, rDNA methylation levels andnucleolar related genes expressionof these embryos. Results1) Nucleolar precursorbodies (NPBs) started to form5h after sperm injection/activation in mouse ICSI andNT embryos. The number of NPBs reduced until8hpa.Then the NPBs number keptunchanging until pronuclei fused at16-17hpa.2) ESNT embryos only formed onepronucleus at pronuclear stage, whiching containing7.20NPBs in average. Cumuluscell nuclear trasnfer (CCNT) rmbryos, MEFs nuclear transfer (MEFNT) embryos,ICSI embryos and parthenogenetic activated (PA) embryos all formed two pronuclei/pseudo-pronuclei and the average NPBs numbers per pronuclei/pseudo-pronucleiwere3.71,4,27,1.58and1.59, respectively.3) The reticular structure started toappear around the nucleoli in ICSI, ESNT and CCNT embryos at late-2cell stage(27hpa). The nucleoli appeard as compeletely reticular structure in ICSI and ESNTembryos at4-cell stage, but nucleoli in CCNT embryos only showed partial reticularstructure at the same stage.4) The CCs lost rRNA synthesis and processing activities after treated with0.8μg/ml actinomycin D for1h. The NT embryos reconstructedfrom AD treated CCs had no significant difference with un-treated group inpreimplantation development competence.5) ESCs had the most active NORs number(7.66) and the lowest rDNA methylation level (6.76%) and its UBF (upstream bindingfactor), FBL (fibrillarin) and B23(nucleophosmin) expression levels weresignificantly higher than CCs and MEFs. MEFs had the least active NORs numbers(4.70) and the highest rDNA methylation levels (22.57%) while CCs had the middleof both (6.45and13.59%).6) At4-cell stage, ESNT embryos had almost the sameactive NORs numbers as ICSI embryos (7.19vs.7.44) and which was significantlyhigher than that in CCNT (6.68) and MEFNT embryos (5.77). MEFNT embryos hadthe highest rDNA methylation level than the other3groups （15.52%vs.9.39%/ICSIvs.6.36%/ESNT vs.9.67%/CCNT）.7) The B23and UBF signals vanished in20minin the nuclei of CCs and MEFs after entering oocyte cytoplasm. But the B23/UBFsignals of ESCs nuclei can also be detected ever at1h after NT.8) Thepreimplantation developmental competence of MEFNT embryos was significantlylower than ICSI, ESNT and CCNT embryos. MEFNT embryos at morula stage hadsignificantly lower FBL and18S rRNA expression levels than the other3groups andtheir UBF and47S rRNA expression levels were significantly lower than ICSIembryos. Conclusion We demonstrated the distribution and changing of nucleolarprotein in donor cells and NT embryos at different preimplantation developnmentalstages. We found these donor cells owned different rDNA activities. And thisdiffenrence determined the different rDNA activities in NT embryos to a large extent,even affected the preimplantation developmental competence of NT embryos.更多还原