本文選題：碎片化 + 植物血凝素��； 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：近年來的干細(xì)胞研究為組織修復(fù)提供了新的治療手段。要把干細(xì)胞有效、安全地應(yīng)用于臨床,必須進(jìn)行深入的機(jī)理研究和大量的在體與離體實(shí)驗(yàn),需要大量的人源干細(xì)胞作為實(shí)驗(yàn)材料。人胚胎干細(xì)胞(human embryonic stem cell, hESC)是從人類早期胚胎內(nèi)細(xì)胞團(tuán)分離出來的高度未分化的全能性細(xì)胞,在體外培養(yǎng)中可保持不分化狀態(tài)而無限增殖,并經(jīng)誘導(dǎo)可分化為不同類型的組織細(xì)胞,因此人胚胎干細(xì)胞建系后可作為理想的實(shí)驗(yàn)材料而用于醫(yī)學(xué)、生物學(xué)、藥學(xué)等方面的研究。 hESC研究的最大制約因素,是胚胎來源有限。在人類的體外受精—胚胎移植(invitro fertilization-embryo transfer, IVF-ET)的過程中,常常會(huì)觀察到產(chǎn)生無核碎片的胚胎。這種有碎片胚胎的發(fā)育潛能受限,移植后很少獲得妊娠,故患者通常會(huì)放棄這種胚胎。這些被放棄的胚胎可以用于人胚胎干細(xì)胞研究。 低評(píng)分的人胚胎經(jīng)去碎片處理后,發(fā)育潛能可以得到明顯提高。表觀遺傳學(xué)狀態(tài)與卵裂球的發(fā)育潛能有關(guān)。H3R26甲基化水平最高的卵裂球會(huì)形成內(nèi)細(xì)胞團(tuán),而甲基化水平低的卵裂球主要形成滋養(yǎng)外胚層。 我們準(zhǔn)備利用碎片超過胚胎體積50%以上的四級(jí)人胚胎,采用酶消化的方法去除胚胎透明帶后,分散卵裂球,去除胚胎碎片,觀察胚胎發(fā)育潛能的變化,并在獲得囊胚后,嘗試進(jìn)行人胚胎干細(xì)胞建系工作。此外,選用增強(qiáng)型綠色熒光蛋白(EGFP)為目的蛋白,觀察體外合成的EGFP-mRNA在小鼠胚胎中的表達(dá)情況,為今后以mRNA顯微注射的方式人為干預(yù)卵裂球發(fā)育潛能打好實(shí)驗(yàn)基礎(chǔ)。 與動(dòng)物克隆的巨大成功相比,人類體細(xì)胞核移植(SCNT)研究的進(jìn)展相當(dāng)緩慢,其主要限制因素是人類卵子受到法律和倫理學(xué)的限制而難以獲得。除了卵子,受精卵的胞質(zhì)也可以支持體細(xì)胞的重編程。在人類IVF中常見的多精受精卵可以作為核移植受體而用于核移植研究,從而解決人類SCNT研究的瓶頸因素。已有研究者利用人多精受精卵進(jìn)行了SCNT研究,但克隆胚胎的發(fā)育沒有超過8細(xì)胞期。 克隆胚胎發(fā)育潛能受限,至少部分原因是重編程不完全。以DNA甲基轉(zhuǎn)移酶抑制劑(如5-氮雜胞苷)來部分擦除已有的表觀遺傳學(xué)標(biāo)記,有利于克隆胚胎的發(fā)育。 我們利用人的多精受精卵為受體,人顆粒細(xì)胞為核供體,嘗試更為簡捷有效的方法進(jìn)行人類體細(xì)胞核移植,并以5-氮雜胞苷對(duì)克隆胚胎進(jìn)行處理,觀察其發(fā)育潛能的變化,驗(yàn)證DNA甲基化水平的降低是否有利于克隆胚胎的發(fā)育。 第一部分人胚胎去碎片培養(yǎng) 方法：(1)去碎片處理。以鏈酶蛋白酶消化人胚胎透明帶,在無鈣鎂培養(yǎng)液中分散卵裂球,以植物血凝素(PHA)處理卵裂球2小時(shí),使卵裂球相互接近,然后對(duì)卵裂球進(jìn)行培養(yǎng),直至形成囊胚。(2)人胚胎干細(xì)胞建系。分離囊胚內(nèi)細(xì)胞團(tuán),進(jìn)行原代克隆培養(yǎng)并連續(xù)傳代。結(jié)果：(1)對(duì)各組人胚胎進(jìn)行培養(yǎng)后,均可達(dá)到囊胚期。對(duì)照組與PHA處理組的囊胚發(fā)育率無差異(分別為30.0%、35%與36.4%),而單純?nèi)ニ槠M的囊胚發(fā)育率明顯降低(11.5%)。(2)對(duì)原代克隆進(jìn)行培養(yǎng)后,繼續(xù)傳代未超過5代,未能獲得人胚胎干細(xì)胞系。結(jié)論：(1)對(duì)低評(píng)分人胚胎進(jìn)行去碎片操作后,能夠獲得高質(zhì)量囊胚。(2)PHA處理對(duì)于分散的卵裂球的發(fā)育有利。 第二部分對(duì)小鼠胚胎進(jìn)行mRNA顯微注射的實(shí)驗(yàn)性研究方法：(1)構(gòu)建pcDNA3.0-EGFP重組質(zhì)粒。(2)體外合成EGFP-mRNA。(3)收集小鼠2細(xì)胞期胚胎,顯微注射EGFP-mRNA,并觀察胚胎發(fā)育情況及熒光表達(dá)情況。結(jié)果：(1)與對(duì)照組相比,接受mRNA(無論是否加polyA尾)顯微注射的小鼠胚胎發(fā)育到囊胚的比率略有下降,但無統(tǒng)計(jì)學(xué)意義(囊胚發(fā)育率：對(duì)照組64.41%；未加polyA尾注射組48.98%；加polyA尾注射組55.74%)。(2)注射未加polyA尾的EGFP-mRNA后,小鼠胚胎沒有表達(dá)綠色熒光；注射加polyA尾的EGFP-mRNA后,小鼠胚胎表達(dá)綠色熒光的比率增加到36.07%,R組與RA組之間表達(dá)熒光的比率差異有統(tǒng)計(jì)學(xué)意義,而囊胚發(fā)育率的差異則無統(tǒng)計(jì)學(xué)意義。結(jié)論：(1) mRNA顯微注射本身并不影響胚胎的發(fā)育潛能。(2) mRNA在細(xì)胞內(nèi)順利表達(dá)的關(guān)鍵是體外合成時(shí)是否成功添加合適長度的polyA尾。第三部分利用人多精受精卵進(jìn)行核移植 方法：實(shí)驗(yàn)分組設(shè)計(jì)如下：PSZ-C:對(duì)照組1,不進(jìn)行任何操作而直接培養(yǎng),以驗(yàn)證培養(yǎng)體系的有效性；PSZ-H:以Hoechst33342進(jìn)行染色后,用紫外線進(jìn)行短暫觀察,以排除核染色與紫外線照射對(duì)胚胎發(fā)育的影響；EO：在間期去除3個(gè)原核中的一個(gè),以驗(yàn)證顯微操作對(duì)胚胎發(fā)育的影響；NT：顆粒細(xì)胞核移植組,未經(jīng)5-氮雜胞苷處理;NT-Aza:顆粒細(xì)胞核移植組,經(jīng)0.01mM5-氮雜胞苷處理。(1)核移植。在有絲分裂間期去除人多精受精卵的細(xì)胞核,以顯微注射方式把人顆粒細(xì)胞的細(xì)胞核注射到去核的受精卵中,按照分組接受或不接受5-氮雜胞苷的處理,進(jìn)行胚胎培養(yǎng)。(2)收集分裂期的人類多精受精卵(PSZ-C)、經(jīng)過5-氮雜胞苷處理的分裂期的核移植胚胎(NT-AZA-C)以及未經(jīng)5-氮雜胞苷處理的分裂期的核移植胚胎(NT-C),進(jìn)行5-甲基胞嘧啶免疫熒光染色,以共聚焦顯微鏡觀察熒光并拍照,以ImageJ軟件分析熒光強(qiáng)度。結(jié)果：(1)顆粒細(xì)胞的細(xì)胞周期分布。72.6+6.0%的顆粒細(xì)胞為G0/G1期。 (2) PSZ-C組單純培養(yǎng)后獲得4個(gè)囊胚,囊胚發(fā)育率為11.1%。PSZ-H組獲得1個(gè)囊胚,囊胚發(fā)育率為7.7%。EO組獲得4個(gè)囊胚,囊胚發(fā)育率為12.9%。各組的8細(xì)胞期胚胎發(fā)育率與囊胚發(fā)育率的差異均無統(tǒng)計(jì)學(xué)意義。(3)核移植的胚胎發(fā)育沒有超過8細(xì)胞期。在NT-CB組中對(duì)29枚胚胎進(jìn)行了操作,存活率為48.3%,卵裂率為21.4%,沒有發(fā)育到8細(xì)胞期的胚胎；在NT組中對(duì)61枚胚胎進(jìn)行了操作,存活率為73.8%,卵裂率為48.9%,發(fā)育達(dá)到8細(xì)胞期的比率為11.1%；在NT-Aza組中對(duì)64枚胚胎進(jìn)行了操作,存活率為81.3%,卵裂率為65.4%,發(fā)育達(dá)到8細(xì)胞期的比率為32.7%。NT-CB組的胚胎存活率、卵裂率和8細(xì)胞期發(fā)育率均低于其余兩組。NT和NT-Aza組在8細(xì)胞期的胚胎發(fā)育率方面的差異具有統(tǒng)計(jì)學(xué)意義。(4) NT-AZA-C和NT-C組之間熒光強(qiáng)度的差異有統(tǒng)計(jì)學(xué)意義,NT-AZA-C組的熒光強(qiáng)度明顯降低,但仍未降至PSZ-C組的水平。(5)核移植胚胎出現(xiàn)異常的核分裂模式。結(jié)論：(1)顆粒細(xì)胞的細(xì)胞周期狀態(tài)適于核移植。(2)本實(shí)驗(yàn)的培養(yǎng)體系條件適宜。(3) Hoechst33342染色及短時(shí)間的紫外線照射不影響胚胎發(fā)育。(4)5-氮雜胞苷處理有利于核移植胚胎的發(fā)育。(5)表觀遺傳學(xué)狀態(tài)的異常與核分裂模式的異常是核移植胚胎發(fā)育潛能受限的重要原因。 小結(jié) 1、對(duì)低評(píng)分人胚胎進(jìn)行去碎片操作后,能夠獲得高質(zhì)量囊胚。PHA處理對(duì)于分散的卵裂球的發(fā)育有利。 2、mRNA顯微注射本身并不影響胚胎的發(fā)育潛能。mRNA在細(xì)胞內(nèi)順利表達(dá)的關(guān)鍵是體外合成時(shí)是否成功添加合適長度的polyA尾。 3、5-氮雜胞苷處理有利于核移植胚胎的發(fā)育。表觀遺傳學(xué)狀態(tài)的異常與核分裂模式的異常是核移植胚胎發(fā)育潛能受限的重要原因。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321

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