Research On Blastula Generation Pathway For Therapeutic Embryonic Stem Cell And Relating Problems

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.