【作者】 武建中；

【导师】 李跃民；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 随着干细胞技术的不断发展,能够用于人类再生医学的具有克服免疫排斥反应的治疗性胚胎干细胞逐渐成为人们研究的重点。通过体细胞核移植技术获得胚胎干细胞,因为具有与体细胞提供者相同的基因型被认为是克服免疫排斥反应的最佳途径,然而,体细胞克隆胚胎由于其重构胚核编程不完全导致其发育率较低,进而影响获取干细胞的成功率;哺乳动物MⅠ期和MⅡ期卵母细胞孤雌激活胚胎与有性生殖胚胎相比,具有与提供卵母细胞的母体相同的组织相容性复合物,由此获得的干细胞能够克服免疫排斥反应。但是来源于MⅡ期卵母细胞孤雌激活胚胎缺乏父源基因印迹作用,其发育潜能受到限制,从而导致其分离获得的干细胞的分化潜能也有可能因此而受到限制:来源于抑制了第一极体排出的MⅠ期卵母细胞孤雌激活胚胎,因为含双倍杂合体基因组核型,能够获得类似于体细胞克隆胚胎的胚胎细胞核型,可在一定程度提高孤雌胚胎的发育潜力并使其分离得到的胚胎干细胞具有正常的细胞核型以用于临床治疗。但是,体细胞克隆胚胎和孤雌胚胎途径都需要囊胚做为胚胎干细胞分离的材料。本试验研究了以上体细胞核移植胚胎和MⅡ期、MⅠ期卵母细胞孤雌激活胚胎三种途径来源囊胚生成的可能性和方法,并研究了影响这三种囊胚发育率的因素,旨在探索昆明小鼠治疗性胚胎干细胞获取的新途径。本研究采用昆明小鼠做为试验动物,针对体细胞克隆胚胎囊胚发育率低,获取其胚胎干细胞困难的问题,选择合适浓度的组蛋白去乙酰化抑制剂（TSA）分别作用于体细胞核移植重构胚的融合和激活阶段,比较了TSA在不同阶段对重构胚发育的影响。旨在通过研究供核体细胞在去核卵母细胞内的核基因重塑修复与重编程过程,分析和探索提高体细胞克隆胚胎囊胚发育率的有效方法。针对孤雌胚胎干细胞获取新途径中,有性生殖胚胎与无性生殖孤雌胚胎嵌合发育,能够在早期胚胎细胞间通过旁分泌与自分泌因素和晚期胚胎发育阶段通过胚胎诱导因素的作用下,促进孤雌胚发育的理论可行性,本研究通过性别鉴定的方法选择雄性有性生殖胚胎与无性生殖MⅡ期卵母细胞孤雌激活胚胎嵌合发育,并可以通过性别鉴定的方法将这种嵌合囊胚分离得到的内细胞团细胞进行来源标记,旨在通过性别鉴定的方法建立有性生殖胚胎辅助与其嵌合发育的无性生殖孤雌胚胎生成干细胞和有效分选分离这种干细胞的新途径。针对孤雌胚胎干细胞获取新途径中,通过抑制体外培养过程中MⅠ期卵母细胞第一极体的排出,获取含双倍体基因组核型的成熟卵母细胞,孤雌激活这种卵母细胞能够获得类似于体细胞克隆胚胎核型的原理,研究MⅠ期卵母细胞双倍体基因组核型获取途径,探讨了小鼠MⅡ期卵母细胞孤雌激活体系是否能成功应用于MⅠ期卵母细胞的孤雌激活,并对不同体外胚胎发育培养液对这种MⅠ期孤雌胚胎的体外培养效果进行了观察,旨在研究MⅠ期卵母细胞双倍体基因组核型人工激活发育为孤雌胚胎的可能性及其早期胚胎体外培养发育的潜力,探索治疗性克隆胚胎孤雌干细胞生成的新途径。试验结果如下:1组蛋白去乙酰化抑制剂（TSA）对体细胞核移植重构胚囊胚生产效果的影响依据体细胞克隆胚胎囊胚生产效率低的原因与供体核染色质和受体卵质之间互作异常、影响体细胞染色质解聚和激活表达程序出现紊乱有关,本试验利用组蛋白去乙酰化酶特异性抑制剂TSA（trychostatin A）能够抑制重构胚早期组蛋白去乙酰化的作用特点,对重构胚发育早期的融合阶段和激活阶段分别或共同进行添加TSA处理,观察其对重构胚发育状况的影响,以揭示组蛋白乙酰化/去乙酰化作用在核内染色质重编程过程中所发挥的作用,旨在探索能够提高体细胞核移植重构胚囊胚生产效率的新途径。结果显示:试验1中处理组3添加50nmol/L TSA作用于核移植重构胚融合和激活阶段（融合3h、激活6h）可以有效抑制组蛋白去乙酰化酶的作用,促进组蛋白的乙酰化,从而达到明显提高重构胚发育的效果,50 nmol/L的TSA浓度处理后的体细胞核移植重构胚的囊胚发育率（14.00%）要显著高于处理组1（0.5nmol/L,5.36%）、处理组2（5 nmol/L,6.12%）、处理组4（500 nmol/L,7.69%）和对照组（0 nmol/L,3.92%）（P＜0.05）;试验2中仅在融合阶段添加TSA抑制组蛋白去乙酰化作用虽然在一定程度能够提高重构胚的囊胚发育率（5.56%）,但与未添加TSA的处理组1（2.63%）相比差异不显著（P＞0.05）,并不能显著改善重构胚的发育状况;试验3中在融合和激活阶段均添加TSA的处理组3和仅在激活阶段添加TSA的处理组2的囊胚发育率（14.71%Vs 12.12%）之间差异不显著,但两者与未添加TSA的对照组的囊胚发育率（6.45%）相比均差异显著（P＜0.05）,可见TSA在激活阶段对供体核染色质组蛋白去乙酰化作用的抑制明显提高了体细胞核移植重构胚的囊胚发育效率;试验4中在融合阶段添加TSA处理时间不变的前提下将TSA在激活阶段的处理时间从6 h延长至9 h,虽然在一定程度上提高了重构胚的囊胚发育率（15.56%）,但与未延长激活时间的处理组1（14.29%）相比囊胚率并没有得到明显的提高,两者之间差异不显著（P＞0.05）,说明延长激活阶段TSA处理时间并不能进一步提高重构胚的发育潜力。2昆白小鼠孤雌胚胎与雄性胚胎嵌合发育研究本研究对小鼠早期胚胎进行性别鉴定,获得雄性胚胎后与孤雌激活胚胎进行嵌合培养,其目的是为了建立一种新的异性胚胎嵌合培养模式,在便于通过分子生物学方法对胚胎不同来源进行性别检测的基础上,研究两种不同类型胚胎细胞之间在嵌合发育过程中的相互影响,为从嵌合发育胚胎中分析标记来源于孤雌胚胎的干细胞研究过程奠定基础。本研究初步建立了构建孤雌激活胚胎与雄性胚胎嵌合胚的方法与程序,对该过程中的几个影响因素做了初步的探讨。结果显示:自主设计的两对性别鉴定引物特异性较强,能够分别扩增出公鼠所特有的250 bp Sry基因片段和公母小鼠共有的399 bp的ZFX基因片段;经过优化的双重PCR体系能够在取2个卵裂球作为PCR反应模板的前提下比较准确的判定小鼠早期胚胎性别:在比较了各种去带液的去带效果及对胚胎的后续发育潜力的影响后发现,在0.5%链蛋白酶液中处理5 min后,胚胎能获得较高的裸胚率（93.33%）和囊胚发育率（76.67%）;应用前面试验建立的小鼠早期胚胎性别鉴定体系鉴定的有性生殖雄性8细胞胚胎与孤雌生殖8细胞胚胎在自制凹窝培养体系的胚胎聚合率（73.91%）与PHA微滴培养体系的胚胎聚合率（62.50%）相比差异显著（P＜0.05）。聚合胚胎在自制凹窝培养体系的囊胚发育率（47.82%）虽然高于PHA微滴培养体系（41.67%）,但两种体系之间差异不显著（P＞0.05）,因此自制凹窝培养体系可以替代PHA微滴培养体系用于胚胎嵌合;通过免疫法对10枚嵌合囊胚内细胞团进行了单细胞分离后有7枚嵌合囊胚的内细胞团单细胞能够在体外增殖。平均每个嵌合囊胚可得到1.40个类ES细胞集落,经对其细胞进行性别鉴定检测后发现,孤雌胚胎来源集落与雄性胚胎来源集落的比例为5:9。3利用小鼠MⅠ期卵母细胞获取孤雌胚胎的研究依据利用细胞松弛素D（CD）能够抑制小鼠MⅠ期卵母细胞排出第一极体,而保持染色体组的倍性,使染色体组类似于体细胞,通过适当的激活方法可使其发育为杂合二倍体孤雌囊胚,为获得与卵母细胞供体组织相容性抗原更加匹配的孤雌生殖胚胎干细胞奠定基础。本研究从激活方法和培养液的选择两个方面对利用CD处理获取二倍体孤雌胚胎进行了初步研究,并且与MⅡ期卵母细胞孤雌胚的发育能力进行了比较。结果显示:在含有5μg/ml浓度CD的mCZB培养基中作用3h,能够成功抑制第一极体的排出;经过乙醇、SrCl₂、A23187、电激活与A23187+6-DMAP五种激活方法激活后胚胎均能发育到囊胚阶段;其中以A23187+6-DMAP激活效果最好（46.15%）,激活后孤雌胚在mCZB培养液中发育到囊胚的比率也最高（19.23%）;应用前面试验确定的最适激活方法处理后的MⅠ期孤雌胚胎分别在mCZB、KSOM、M16和mM16四种培养液中培养后发现,以mM16培养液中的MⅠ期孤雌囊胚发育率最高（16.67%）;应用A23187+6-DMAP分别对MⅠ和MⅡ期卵母细胞激活后培养于mM16培养液中发现,MⅡ期卵母细胞的卵裂率显著高于MⅠ期卵母细胞（65.88%Vs46.15%,p＜0.05）,但两者的囊胚发育率之间差异不显著（18.82%Vs 17.58%,p＞0.05）。以上三个试验,观察探索了用于分离昆明小鼠治疗性胚胎干细胞的囊胚生成的新途径;综合对不同来源囊胚生成途径的关键问题进行了研究。研究结论如下:1.在昆白小鼠体细胞克隆胚胎的制作过程中,重构胚于激活阶段,在添加有50 nmol/LTSA的KSOM激活培养基中作用6 h,可以在最小程度伤害重构胚的情况下最大限度的实现对组蛋白去乙酰化酶的抑制,促进组蛋白的乙酰化,可以明显提高重构胚发育至囊胚的效率。2.双引物双重PCR法能够成功的对小鼠早期胚胎进行性别鉴定;应用该方法选择出的雄性胚胎与孤雌胚胎在自制凹窝培养体系中可成功发育至囊胚;同时应用这种性别鉴定方法,还成功的对嵌合囊胚内细胞团单细胞培养增殖的类ES细胞集落的来源进行了检测;从而成功建立了一种新的利用有性生殖胚胎辅助与其嵌合发育的无性生殖孤雌胚胎生成干细胞和有效分选分离这种干细胞的新的研究模型。3.昆白小鼠MⅠ期卵母细胞在CD处理后经A23187和6-DMAP激活后可成功获得二倍体孤雌胚胎并发育至囊胚;其囊胚发育率与MⅡ期卵母细胞孤雌激活胚胎相比差异不显著。更多还原

【Abstract】 Therapeutic cloning embryonic stem cell gradually become the focal point due to its overcoming immunological rejection,as unceasing development.of stem cell technology.To obtain embryonic stem cell by implantation technique,because of the same genetype as somatocyte supplier is considered as the best method for overcoming immunological rejection.However,the isolation rate of stem cell from cloning embryo is very low because of its low development caused by incomple programming of reconstructed embryo.MⅠand MⅡpartheno-embryo possessed the same MHC as oocyte supplier,so the stem cell obtained from which can overcome immune rejection,compared with sexual embryo.But the development potential and the differentiation abilities of stem cell isolated from MⅡpartheno-embryo are limited because of paternal printing-gene absence.Sexual male embryo jogged with partheno-embryo from MⅡoocyte to form chimeric embryo which can promote the development of partheno-embryo theoretically because of paracrine and autocrine factors between cell sand embryonic induction effects.The kind of stem cell from this method can provide a new pathway for therapeutic embryonic stem cell.Parthenoembryo from MⅠoocyte which Pb1 was inhibited to discharge,can be used to isolate the stem cell with the normal karyotype for clinic applications.But the blatula is the common material for stem cell isolation.In this experiment,the possibilities and new methods of blastula generations from MⅠ, MⅡpartheno-embryos and somatic cloning embryo were researched,aiming to explore the new pathway for mouse therapeutic embryonic stem cell.In this study,KM mice as experimental animals,due to the low development rate of somatic cloning embryo and low isolation rate of stem cell,we selected the appropriate concentrations of histone deacetylase inhibitor(TSA),respectively,acting on somatic cell nuclear transfer embryos at fuse and activation stages,compared the impact of TSA treated at different stages of embryo development on reconstructed embryo.We use mouse embryonic fibroblast as feeder layer to isolate embryonic stem cells with the whole embryo and immune serum methods in order to observe its development potential.Through researching for the gene repair and remodeling process of re-programming in somatic cell nuclear,we analysis the new pathway to increase the development rate of blastocysts;by studying the separation of stem cell from cloned blastocysts we explore the effective method to isolate stem cell from somatic cloning embryo.As we know,the chimeric embryo from sexual male embryo and partheno-embryo can promote the normal development of partheno-embryo by paracrine and autocrine factors in late stages of embryonic development.In this study,we research the chimeric embryo development and isolate the stem cell from chimeric blastula to establish a new method for stem cell isolation from this chimeric embryo with sexing means.In the new method for parthenogenetic stem cell,we obtained mature oocytes with diploid karyotype by inhibiting the Pb1 discharge and activated this oocyte to obtain tha same karyotype with somatic cloning embryo.According to this principle,we explored the diploid karyotype access of MⅠstage oocytes,compared the impacts of parthenogenetic activation system on MⅠoocytes parthenogenetic activation effects and observed the development of MⅠparthenogenetic embryos in vitro treated different culture media,in order to the possibility of parthenogenetic embryo from MⅠoocyte with diploid karyotype and the development potential of early embryo in vitro,to explore the new pathway for therapeutic stem cell.Results as follows:1.The impact of Histone Deacetylase inhibitors(TSA) on the blastula production of reconstructed embryo from nuclear transplantationAccording to the low development rate of somaic cloning embryo is related with the disorder of somatochrome depolymerization and activation procedure caused by the abnormal interaction between donor chromatin and receptor ooplasm,on fuse and activation stages,we treated with TSA respectively to observe the impact of TSA on reconstructed embryo,in order to reveal the effects of acetylation and deacetylation of histone on chromatin reprogramming process,exploring a new pathway to improve the development rate of nuclear cloning reconstructed embryo.Results: Addition of 50 nmol/L TSA at the stage of activation and fusion for reconstructed embryo cultured in KSOM medium for 9h(fusion:3h,activation:6h),can promote the acetylation of histone effectively and improve the development of reconstructed embryo.The development rate after 50 nmol/L TSA treated(14.00%) was significantly higher than other four groups(0.5 nmol/L,5.36%;5 nmol/L,6.12%;500 nmol/L,7.69%;0 nmol/L,3.92%;P＜0.05);But addition TSA at the fuse stage had no significant difference with control group(5.26%Vs 2.63%,P＞0.05).By inhibiting histone deacetylation at the activation stage,the reconstructed embryo had higher development rate.Group 2 and group 3 added TSA had significantly higher rate than other groups not added TSA(14.71%, 12.12%Vs 6.45%,P＜0.05).In the fourth experiment,we extend the processing time of the activation staged(from 6h to 9h),However,extending the handing time of TSA at the activation stage can’t improve the development conditions of reconstructed embryo(15.56%Vs14.29%, P＞0.05).2.Research on chimeric blastula of parthenogenetic embryo and sexual male embryoIn this experiment,we established a new chimeric culture patternof heterology embryo by choosing sexual male embryo with sexing method jogged with parthno-embryo to research the interaction of this two kinds of embryos in chimeric development and set basis for analysis labeling parthnogenetic stem cell.This study established the methods and procedure of chimeric embryo construction and explored the relative problems in this process.Results:Two pairs of sexing primers desighed independently had stronger specificity and can amplify 250 bp Sry gene typical for male mouse and 399 bp ZFX gene for female mouse.Optimized PCR system can determine the sexuality of early embryo by the premise of 2 blastomeres as PCR template.It was found that the embryo had higher blastula rate(76.67%) and higher gymnoblast rate(93.33%) after 0.5% protease treated.Two culture methods were compared according as the development rate of aggregated embryos and the blastocyst rate.The results showed that the aggregated embryos cultured in hollowness(47.82%) had appreciably higher embryo development rate and blastocyst rate than that of these cultured in culture dish with PHA(41.67%),but there was no significant difference between them(P＞0.05).So hollowness culture system self-made can substitude PHA droplet system for chimeric research.In the process of partheno-embryo developing to blatula.there are 7 chimeric embryos can proliferation in vitro by immunifaction from 10 chimeric embryos,and every chimeric embryo can form 1.40 ES cell colony on average.Through Sexing detection,we found that the ratio between cell colony derived from sexual male embryo and chimeric embryo was 5:9.3.Research on parthenogenetic embryo from MⅠimmature oocyteCB can make chromosome keep the normal karytype by inhibiting Pb1 dischage to overcome gene imprinting problems.And parthenogenetic individual from female germ cell was expected to obtain by artificial intervention.In this experiment,we researched diploid partheno-embryo treated with CD and compared the development abilities with MⅡoocyte.Results:CD can inhibit Pb1 discharge effectively for MⅠoocyte.A23187+6-DMAP was the best activation method for MⅠoocyte with Pb1(46.15%),and the blastula development rate was higher than other methods(19.23%);Of mCZB,KSOM,M16和mM16 the four media,mM16 was the best culture medium for MⅠblastula development(16.67%).MⅠoocyte activated partheno-embryo had no significantly higher blastula rate than MⅡphase(18.82%Vs 17.58%,p＞0.05).Three experiments above,explored the new blastula generation pathway for therapeutic stem cell and analyzed the factors influenced the blastula development.Some important problems in the pathway of blastula generation were researched in this experiment.Conclusions as follows:1 Reconstructed embryo was cultured in KSOM medium added 50 nmol/L TSA for 6 h at the activation stage can promote histone acetylation utmost with the minimum impact for embryo and can improve the blastula development rate obviously.2 Double pairs primers dual PCR for sex identification system is an optimize the sex identification system.The sexual male embryos which were chosen by this method can development to the blastula with the parthenogenetic embryo in in hollowness;Meanwhile,the sex identification system also can detect the source from the cell colony.3 CD can inhibit Pb1 discharge effectively for MⅠoocyte,A23187+6-DMAP was the best activation method for MⅠoocyte with Pb1,and MⅠoocyte activated partheno-embryo had no significantly higher blastula rate than MⅡphase.更多还原