【作者】 梁素丽；

【导师】 靳亚平；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 博士

【摘要】 体细胞核移植是将高度分化的体细胞移入去核的卵母细胞内,成功的体细胞核移植需要在很短的时间内消除原有的供体细胞的分化记忆,将供体细胞核重编程为全能的、胚胎化的状态,并接受卵胞质内的信息,表现出一种新的与胚胎发育密切相关的基因表达模式,这其中包括了复杂的表观遗传的改变。已分化的供体细胞在很短的时间内很难完成如此大量的工作,因此也就造成了核移植效率的低下。本文以研究牛体细胞核移植重构胚的重编程为目标,进行了Ca²⁺在牛卵母细胞成熟、体外受精（IVF）、孤雌激活（PA）及核移植（SCNT）胚胎发育过程中的分布规律及其动态变化的研究;探讨了MG-132对重构胚重编程的作用,研究了核纤层蛋白（lamin A/C）在PA、IVF和NT胚胎的表达情况,对比了Oct-4在SCNT和IVF胚胎发育过程中的表达变化。1.南阳牛耳源组织成纤维细胞的培养?以南阳牛耳缘组织为研究材料,体外培养了成纤维细胞,并对其进行了形态学、细胞生长动力学观察、染色体核型和乳酸脱氢酶、苹果酸脱氢酶的同工酶分析及,作为后续研究的实验材料。2.南阳牛卵母细胞成熟和早期胚胎发育过程中Ca²⁺分布规律及其动态变化?采用激光扫描共聚焦显微镜对南阳牛卵母细胞成熟、IVF、PA及SCNT胚胎发育过程中Ca²⁺分布规律及其动态变化进行了全面的研究。Ca²⁺在GV期主要分布在卵皮质区域;GVBD期开始向细胞中心扩散;MⅠ期到MII期Ca²⁺均匀分布于整个细胞。Ca²⁺浓度从GV期开始不断上升,MⅠ期时达到最大,随后开始下降。IVF胚胎原核形成前Ca²⁺主要分布在卵皮质区域;原核形成后,Ca²⁺在整个细胞中都分布;随后Ca²⁺均匀分布于整个卵裂球中,至到囊胚期Ca²⁺明显降低,且分布不均匀,Ca²⁺荧光强度有明显的规律性变化;PA胚胎0²4 h Ca²⁺主要分布在卵皮质区;48 h左右时Ca²⁺在整个卵裂球中都有分布,在核区荧光强度稍强;4-细胞、8-细胞、桑葚胚时,Ca²⁺均匀分布于卵裂球中;囊胚时Ca²⁺分布不均匀;整个过程中Ca²⁺荧光强度变化没有明显规律;核移植24 h后Ca²⁺分布于供体细胞并均匀分布于胞质中;2-细胞、4-细胞、8-细胞中Ca²⁺均匀分布于各卵裂球中;桑葚胚和囊胚期Ca²⁺分布不均匀。整个过程Ca²⁺荧光强度变化呈明显的规律性变化。3. MG-132对牛SCNT重构胚重编程的影响?通过MG-132作用于SCNT全过程所得重构胚的发育情况,研究MG-132的作用,并通过免疫组化方法进行验证。5μmol/mL MG-132作用于MI期卵母细胞可有效阻止MPF活性的下降。未添加MG-132组,融合4 h后lamin A/C着色显著,说明染色体凝集不完全,添加组融合4 h后,核膜不完全着色,明显发生了染色体凝集。说明5μmol/mL MG-132就能够促进重构胚的重编程。在SCNT整个过程中添加5μmol/mL MG-132,通过对比lamin A/C在孤雌激活、IVF和核移植重构胚的表达,首先确定lamin A/C可以作为表征重编程的一个因素,其次lamin A/C在核移植重构胚的早期胚胎发育中几乎不表达,8-细胞期开始表达,囊胚期滋养层细胞中正常表达,但内细胞团中表达减弱,这一模式同IVF胚胎的表达模式相近,进一步证明MG-132的添加能够促进重构胚的重编程。4. Oct-4在SCNT重构胚中的表达应用激光扫描共聚焦显微镜研究显示,添加MG-132后,Oct-4在2-4细胞期几乎不表达,到8-16细胞期开始表达;到桑葚胚期和致密桑葚胚期Oct-4强表达;囊胚期Oct-4在内细胞团和滋养层细胞中都持续强表达;孵化囊胚期Oct-4的表达同囊胚期,并未见滋养层细胞中表达明显减弱,这一结果与IVF胚胎中Oct-4起始表达相同,但孵化囊胚期的表达略有不同。以上研究分析了Ca²⁺在牛卵母细胞成熟、IVF、PA和SCNT胚胎中的分布规律及其动态变化,有望通过进一步研究提高核移植激活效率,最终达到提高核移植效率的目的;lamin A/C可以作为表征核移植重编程的一个因素,MG-132用于核移植过程,并对重构胚的重编程具有促进作用,添加MG-132后Oct-4的起始表达与IVF胚胎相同。通过上述研究有望提高核移植效率。更多还原

【Abstract】 Somatic cell nuclear transfer （SCNT） requires cytoplast-mediated reprogramming of the donor nucleus. Successful SCNT requires erasing the inherent differentiation memory or program, present in differentiated donor nuclei and reprogram dedifferentiated state of nucleus to development-specific totipotent pattern like the normal embryos. All these changes refer to remodeling. Differentiated donor cells were difficult to accomplish such a lot of work in a short period, so it was one of the reasons of low SCNT efficiency. In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization （IVF）, parthenogenetic activation （PA） and SCNT embryo development. To investigate the effect of MG-132 on the nuclear remodeling of donor cells after transferring into bovine oocytes.1. The establishment of an ear marginal fibroblast cell from Nanyang bovineAn ear marginal fibroblast cell was established from Nanyang bovine using a primary explant technique and cell cryopreservation biotechniques. Cell morpHology, dynamic growth and contamination were tested, and the karyotype, levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed.2. Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in IVF, PA and SCNT embryo development.In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, IVF, PA and SCNT embryo development. During the germinal vesicle （GV） and GV breakdown stage, Ca²⁺ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca²⁺ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca²⁺ was present throughout the blastomere. In PA embryos, Ca²⁺ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell pHase and the morula. At 6 h after activation, there was almost no distribution of Ca²⁺ in the SCNT embryos. However, Ca²⁺ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca²⁺ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca²⁺ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.3. The effect of MG-132 on the nuclear remodelingTo investigate the effect of MG-132 on the nuclear remodeling of donor cells after transferring into bovine oocytes. We studied the role of MG-132 by examination the developmental potential of reconstructed embryos that got from nuclear transfer. The entire process of nuclear transfer was acting by MG-132, we verify the results by immunofluorescence. The reconstructed embryos 4 h after fusion showed strong lamin staining of the control group. The group of adding MG-132 at 4h after fusion there showed patchy nuclear membrance attaining and significant chromatin condensation. The results indicate that adding of 5μmol/mL MG-132 in SCNT might promote the reprogramming of reconstructed embryos. Then 5μmol/mL MG-132 was addedn in nuclear transfer operation. We compared the expression of lamin A/C in PA、IVF and SCNT embryos. The results indicate that lamin A/C is an indicator of erroneous reprogramming. Lamin A/C signal was negative in early reconstructed embryos, expressed in 8-cell stage. Lamin A/C was expressed in tropHectodermal cells of blastocysts, but signal was diminished in ICM. This is same to IVF embryos of lamin A/C expression. It proved that adding of MG-132 can improve reprogramming. In SCNT embryos, no lamin A/C was expressed in 4-cell stage. It began to express in 8-cell stag; Lamin A/C was expressed in 16-cell stage, morula stage and blastocysts. Signal was expressed in trophectodermal cells and ICM of blastocysts. But in IVF embryos, Lamin A/C was expressed in trophectodermal cells of blastocysts, but signal was diminished in ICM. It need further study to define the different expression in blastocysts effecton the embryonic development.4. The expressiong of Oct-4 in IVF and SCNT embryosOct-4 was almost no expressed in 2-4 cell phase, and expressed from 8-16 cell stage. Strong oct-4 signal was expressed in morula and blastocysts. The expression of oct-4 in hatching blastocyst stage was same to hatching blastocyst stage, but it was not decreased in trophoblast cells. The initial expression of SCNT oocytes was same to IVF, but the expression was not decreased in trophoblast cells in IVF oocytes. If this different expression will affect the development of post-implantation embryo, it remains to be study. This rearcher was investigated the distribution pattern of Ca2+ and its dynamic changes in the processes of bovine oocytes maturation, IVF, PA and SCNT embryos development, further research is expected to improve the SCNT efficiency. The research of lamin A/C expression showed that MG132 could be used for nuclear transfer process, and could improve the reprogramming of reconstructed embryos, and the expression of Oct-4 was same to IVF embryos.更多还原