提高人低评分胚胎发育潜能及利用人多精受精卵进行核移植的实验性研究

发布时间：2018-04-23 04:18

本文选题：碎片化 + 植物血凝素　；参考：《第二军医大学》2012年博士论文

【摘要】：近年来的干细胞研究为组织修复提供了新的治疗手段。要把干细胞有效、安全地应用于临床,必须进行深入的机理研究和大量的在体与离体实验,需要大量的人源干细胞作为实验材料。人胚胎干细胞(human embryonic stem cell, hESC)是从人类早期胚胎内细胞团分离出来的高度未分化的全能性细胞,在体外培养中可保持不分化状态而无限增殖,并经诱导可分化为不同类型的组织细胞,因此人胚胎干细胞建系后可作为理想的实验材料而用于医学、生物学、药学等方面的研究。 hESC研究的最大制约因素,是胚胎来源有限。在人类的体外受精—胚胎移植(invitro fertilization-embryo transfer, IVF-ET)的过程中,常常会观察到产生无核碎片的胚胎。这种有碎片胚胎的发育潜能受限,移植后很少获得妊娠,故患者通常会放弃这种胚胎。这些被放弃的胚胎可以用于人胚胎干细胞研究。低评分的人胚胎经去碎片处理后,发育潜能可以得到明显提高。表观遗传学状态与卵裂球的发育潜能有关。H3R26甲基化水平最高的卵裂球会形成内细胞团,而甲基化水平低的卵裂球主要形成滋养外胚层。我们准备利用碎片超过胚胎体积50%以上的四级人胚胎,采用酶消化的方法去除胚胎透明带后,分散卵裂球,去除胚胎碎片,观察胚胎发育潜能的变化,并在获得囊胚后,尝试进行人胚胎干细胞建系工作。此外,选用增强型绿色荧光蛋白(EGFP)为目的蛋白,观察体外合成的EGFP-mRNA在小鼠胚胎中的表达情况,为今后以mRNA显微注射的方式人为干预卵裂球发育潜能打好实验基础。与动物克隆的巨大成功相比,人类体细胞核移植(SCNT)研究的进展相当缓慢,其主要限制因素是人类卵子受到法律和伦理学的限制而难以获得。除了卵子,受精卵的胞质也可以支持体细胞的重编程。在人类IVF中常见的多精受精卵可以作为核移植受体而用于核移植研究,从而解决人类SCNT研究的瓶颈因素。已有研究者利用人多精受精卵进行了SCNT研究,但克隆胚胎的发育没有超过8细胞期。克隆胚胎发育潜能受限,至少部分原因是重编程不完全。以DNA甲基转移酶抑制剂(如5-氮杂胞苷)来部分擦除已有的表观遗传学标记,有利于克隆胚胎的发育。我们利用人的多精受精卵为受体,人颗粒细胞为核供体,尝试更为简捷有效的方法进行人类体细胞核移植,并以5-氮杂胞苷对克隆胚胎进行处理,观察其发育潜能的变化,验证DNA甲基化水平的降低是否有利于克隆胚胎的发育。第一部分人胚胎去碎片培养方法：(1)去碎片处理。以链酶蛋白酶消化人胚胎透明带,在无钙镁培养液中分散卵裂球,以植物血凝素(PHA)处理卵裂球2小时,使卵裂球相互接近,然后对卵裂球进行培养,直至形成囊胚。(2)人胚胎干细胞建系。分离囊胚内细胞团,进行原代克隆培养并连续传代。结果：(1)对各组人胚胎进行培养后,均可达到囊胚期。对照组与PHA处理组的囊胚发育率无差异(分别为30.0%、35%与36.4%),而单纯去碎片组的囊胚发育率明显降低(11.5%)。(2)对原代克隆进行培养后,继续传代未超过5代,未能获得人胚胎干细胞系。结论：(1)对低评分人胚胎进行去碎片操作后,能够获得高质量囊胚。(2)PHA处理对于分散的卵裂球的发育有利。第二部分对小鼠胚胎进行mRNA显微注射的实验性研究方法：(1)构建pcDNA3.0-EGFP重组质粒。(2)体外合成EGFP-mRNA。(3)收集小鼠2细胞期胚胎,显微注射EGFP-mRNA,并观察胚胎发育情况及荧光表达情况。结果：(1)与对照组相比,接受mRNA(无论是否加polyA尾)显微注射的小鼠胚胎发育到囊胚的比率略有下降,但无统计学意义(囊胚发育率：对照组64.41%；未加polyA尾注射组48.98%；加polyA尾注射组55.74%)。(2)注射未加polyA尾的EGFP-mRNA后,小鼠胚胎没有表达绿色荧光；注射加polyA尾的EGFP-mRNA后,小鼠胚胎表达绿色荧光的比率增加到36.07%,R组与RA组之间表达荧光的比率差异有统计学意义,而囊胚发育率的差异则无统计学意义。结论：(1) mRNA显微注射本身并不影响胚胎的发育潜能。(2) mRNA在细胞内顺利表达的关键是体外合成时是否成功添加合适长度的polyA尾。第三部分利用人多精受精卵进行核移植方法：实验分组设计如下：PSZ-C:对照组1,不进行任何操作而直接培养,以验证培养体系的有效性；PSZ-H:以Hoechst33342进行染色后,用紫外线进行短暂观察,以排除核染色与紫外线照射对胚胎发育的影响；EO：在间期去除3个原核中的一个,以验证显微操作对胚胎发育的影响；NT：颗粒细胞核移植组,未经5-氮杂胞苷处理;NT-Aza:颗粒细胞核移植组,经0.01mM5-氮杂胞苷处理。(1)核移植。在有丝分裂间期去除人多精受精卵的细胞核,以显微注射方式把人颗粒细胞的细胞核注射到去核的受精卵中,按照分组接受或不接受5-氮杂胞苷的处理,进行胚胎培养。(2)收集分裂期的人类多精受精卵(PSZ-C)、经过5-氮杂胞苷处理的分裂期的核移植胚胎(NT-AZA-C)以及未经5-氮杂胞苷处理的分裂期的核移植胚胎(NT-C),进行5-甲基胞嘧啶免疫荧光染色,以共聚焦显微镜观察荧光并拍照,以ImageJ软件分析荧光强度。结果：(1)颗粒细胞的细胞周期分布。72.6+6.0%的颗粒细胞为G0/G1期。 (2) PSZ-C组单纯培养后获得4个囊胚,囊胚发育率为11.1%。PSZ-H组获得1个囊胚,囊胚发育率为7.7%。EO组获得4个囊胚,囊胚发育率为12.9%。各组的8细胞期胚胎发育率与囊胚发育率的差异均无统计学意义。(3)核移植的胚胎发育没有超过8细胞期。在NT-CB组中对29枚胚胎进行了操作,存活率为48.3%,卵裂率为21.4%,没有发育到8细胞期的胚胎；在NT组中对61枚胚胎进行了操作,存活率为73.8%,卵裂率为48.9%,发育达到8细胞期的比率为11.1%；在NT-Aza组中对64枚胚胎进行了操作,存活率为81.3%,卵裂率为65.4%,发育达到8细胞期的比率为32.7%。NT-CB组的胚胎存活率、卵裂率和8细胞期发育率均低于其余两组。NT和NT-Aza组在8细胞期的胚胎发育率方面的差异具有统计学意义。(4) NT-AZA-C和NT-C组之间荧光强度的差异有统计学意义,NT-AZA-C组的荧光强度明显降低,但仍未降至PSZ-C组的水平。(5)核移植胚胎出现异常的核分裂模式。结论：(1)颗粒细胞的细胞周期状态适于核移植。(2)本实验的培养体系条件适宜。(3) Hoechst33342染色及短时间的紫外线照射不影响胚胎发育。(4)5-氮杂胞苷处理有利于核移植胚胎的发育。(5)表观遗传学状态的异常与核分裂模式的异常是核移植胚胎发育潜能受限的重要原因。小结 1、对低评分人胚胎进行去碎片操作后,能够获得高质量囊胚。PHA处理对于分散的卵裂球的发育有利。 2、mRNA显微注射本身并不影响胚胎的发育潜能。mRNA在细胞内顺利表达的关键是体外合成时是否成功添加合适长度的polyA尾。 3、5-氮杂胞苷处理有利于核移植胚胎的发育。表观遗传学状态的异常与核分裂模式的异常是核移植胚胎发育潜能受限的重要原因。
[Abstract]:In recent years, stem cell research has provided a new therapeutic method for tissue repair. To apply stem cells effectively and safely to clinical trials, it is necessary to conduct in-depth mechanism research and a large number of in vivo and in vitro experiments. A large number of human stem cells are needed as experimental materials. Human embryonic stem cells (human embryonic stem cell, hESC) are from human early The highly undifferentiated omnipotent cells isolated from the cell masses in the embryonic stage can remain undifferentiated and proliferate in vitro, and can be differentiated into different types of tissue cells. Therefore, human embryonic stem cells can be used as ideal experimental materials for medical, biological and pharmaceutical research.
The biggest constraint in hESC research is the limited source of embryos. In the process of human invitro fertilization-embryo transfer (IVF-ET), embryos are often observed to produce nuclear free fragments. The developmental potential of the fragmented embryos is limited and the pregnancy is rarely obtained after transplantation, so patients usually give up this These aborted embryos can be used for human embryonic stem cell research.
The developmental potential of the low grade human embryo can be significantly improved. The epigenetic status and the developmental potential of the blastomere are related to the inner cell mass of the blastomere with the highest level of.H3R26 methylation, while the low level of the blastomere mainly forms the nourishing ectoderm.
We are going to use the four level human embryo with more than 50% of the volume of the embryo. We use the enzyme digestion method to remove the zona pellucida, disperse the blastomere, remove the embryo fragments and observe the changes of the developmental potential of the embryo. After obtaining the blastocyst, we try to build the human embryonic stem cell line work. In addition, the enhanced green fluorescent protein (EGFP) is selected. For the purpose of protein, the expression of EGFP-mRNA in mouse embryos was observed in vitro, and the experimental basis for the human intervention of blastomere development potential by mRNA microinjection in the future.
Compared with the great success of animal cloning, the progress of human nuclear transfer (SCNT) research is rather slow. The main limiting factor is that human eggs are difficult to obtain by legal and ethical limitations. In addition to eggs, the cytoplasm of fertilized eggs can also support the recompilation process of somatic cells. The multiple sperm fertilized eggs common in human IVF can be used as a result. Nuclear transplant recipients are used in nuclear transplantation research to solve the bottleneck factors in human SCNT research. Researchers have used human sperm fertilized eggs for SCNT research, but the development of cloned embryos is not more than 8 cell stages.
The developmental potential of cloned embryos is limited, at least partly because of incomplete reprogramming. DNA methyltransferase inhibitors (such as 5- aza cytidine) are used to erase the existing epigenetic markers, which are beneficial to the development of cloned embryos.
We use human sperm cells as the receptor and human granulosa cells as nuclear donors to try more simple and effective methods to carry out human somatic cell nuclear transplantation, and use 5- aza cytidine to treat cloned embryos, observe the changes of their developmental potential, and verify whether the lower level of DNA methylation is beneficial to the development of cloned embryos.
The first part of human embryo to fragment culture
Methods: (1) defragment treatment. Use chain enzyme protease to digest the human embryo clear zone, disperse blastomere in no calcium magnesium medium, treat blastomere with plant hemagglutinin (PHA) for 2 hours, make blastomere close to each other, and then culture blastomere until the blastocyst is formed. (2) human embryonic stem cells are built. The cell mass in blastocyst is separated and original generation is carried out. Cloned culture and continuous subculture. Results: (1) the blastocyst period can be reached after culture of each group of human embryos. The development rate of blastocyst in the control group and the PHA treatment group is not different (30%, 35% and 36.4% respectively), while the blastocyst development rate of the single fragment group is significantly lower (11.5%). (2) after the culture of the primary clones, the growth rate of the blastocyst is not more than 5 generations. The human embryonic stem cell line can be obtained. Conclusion: (1) high quality blastocysts can be obtained after the removal of the low score human embryo. (2) PHA treatment is beneficial to the development of the dispersed blastomeres.
The second part of the second part of the experimental study of mouse embryos microinjection: (1) construction of pcDNA3.0-EGFP recombinant plasmid. (2) to synthesize EGFP-mRNA. (3) in vitro to collect mouse 2 cell stage embryos, microinjection of EGFP-mRNA, and observe the development of embryo and fluorescence expression. (1) compared with the control group, accept mRNA (whether or not POL) YA tail) the rate of mouse embryo development to blastocyst decreased slightly, but there was no statistical significance (blastocyst development rate: control group 64.41%, no polyA tail injection group 48.98%, and polyA tail injection group 55.74%). (2) after injection of EGFP-mRNA without polyA tail, mice embryos did not express green fluorescence; after injection of polyA tail EGFP-mRNA, The ratio of green fluorescence in mouse embryos increased to 36.07%, and there was significant difference in the ratio of expression of fluorescence between R and RA groups, but there was no significant difference in the development rate of blastocysts. (1) mRNA microinjection itself did not affect the developmental potential of the embryo. (2) the key to the smooth expression of mRNA in the cells was in vitro synthesis. No suitable length of polyA tail was successfully added. The third part used human sperm fertilized eggs for nuclear transfer.
Methods: the experimental group was designed as follows: PSZ-C: control group 1, without any operation and direct culture, to verify the effectiveness of the culture system; PSZ-H: was stained with Hoechst33342 and observed briefly with ultraviolet light to eliminate the effects of nuclear and ultraviolet radiation on the development of embryos; EO: one of the 3 prokaryotes was removed at intervals. To verify the effect of micromanipulation on the development of embryos; NT: granular cell nuclear transplantation group, without 5- heterocytidine treatment, NT-Aza: granular cell nuclear transplantation group, 0.01mM5- aza cytidine treatment. (1) nuclear transplantation. The nucleus of the human sperm fertilized eggs was removed at the mitosis interval, and the nuclei of human granulosa cells were injected into the cell nucleus by microinjection. In the fertilized egg of the nucleus, the embryo culture was carried out according to the group acceptance or non acceptance of 5- nitrocytidin. (2) the mitotic human sperm (PSZ-C) was collected at the split stage, the nuclear transplant embryo (NT-AZA-C), which was treated by 5- heterocytidine, and the nuclear transplanting embryo (NT-C), which was not treated by the 5- nitrocytidine, was used to carry out 5- methyl cells. Pyrimidine immunofluorescence staining was used to observe fluorescence and photograph by confocal microscopy. The fluorescence intensity was analyzed with ImageJ software. Results: (1) the cell cycle distribution of granular cells in.72.6+6.0% was G0/G1 phase.
(2) in group PSZ-C, 4 blastocysts were obtained after simple culture, and the development rate of blastocyst was 1 blastocysts in group 11.1%.PSZ-H. The development rate of blastocyst was 4 blastocysts in group 7.7%.EO. There was no significant difference in the difference of the 8 cell stage embryo development rate between the blastocyst development rate and the blastocyst development rate in each group of 12.9%.. (3) the embryonic development of nuclear transplantation did not exceed the 8 cell stage. In N In group T-CB, 29 embryos were operated, the survival rate was 48.3%, the cleavage rate was 21.4%, the 8 cell stage embryos were not developed; the 61 embryos were operated in the group NT, the survival rate was 73.8%, the cleavage rate was 48.9%, the rate of 8 cell stage was 11.1%, and 64 embryos were operated in group NT-Aza, the survival rate was 81.3%, egg survival rate. The ratio of the split rate was 65.4%, the ratio of the 8 cell stage to the 32.7%.NT-CB group was the embryo survival rate, the cleavage rate and the 8 cell stage development rate were lower than those of the other two groups of.NT and NT-Aza group in the 8 cell stage. (4) the difference of fluorescence intensity between NT-AZA-C and NT-C group was statistically significant, NT-AZA-C group was statistically significant. The fluorescence intensity decreased obviously, but still did not decrease to the level of PSZ-C group. (5) the abnormal nuclear split pattern in the nuclear transplanted embryos. Conclusion: (1) the cell cycle status of granular cells is suitable for nuclear transplantation. (2) the conditions of this experiment are suitable. (3) Hoechst33342 staining and short time ultraviolet radiation do not affect embryo development. (4) 5- heterocytidine Treatment is beneficial to the development of nuclear transplant embryos. (5) abnormality in epigenetic state and abnormality of the mode of nuclear mitosis are important reasons for limited development potential of nuclear transfer embryos.
Summary
1, after the fragmentation of the low score human embryos, high quality blastocyst.PHA treatment is beneficial to the development of the blastomere.
2, mRNA microinjection itself does not affect the development potential of the embryo, the key to the successful expression of.MRNA in the cell is whether the suitable length of polyA tail is successfully added when in vitro synthesis.
3,5- aza cytidine treatment is beneficial to the development of nuclear transplanted embryos. Abnormality in epigenetic state and abnormality of the mode of nuclear mitosis are important reasons for the limitation of the developmental potential of nuclear transplant embryos.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321

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