The Study On Influencing Factors Of Chimeric Parthenogenetic Embryos Development And Different Sex Rate About The ICM Of The Chimeric Parthenogenetic Blastula

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 stern cell from cloning embryo is very low because of its low development caused by incomple programming of reconstructed embryo.But parthenogenetic stem cell is obtained from germ cell which still not experienced cleavage,proliferation and differentiation.So this kind of stem cell doesn't contain mutable genes of suffering animals in theories.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 blastocyst inner cell mass from this method can provide a new pathway for therapeutic embryonic stem cell.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 blastocyst inner cell mass from chimeric blastula to establish a new method for stem cell isolation from this chimeric embryo with sexing means.Experiment summarized as follows:1 The Study of cultural method and effect on mouse embryonic chimeras between Male and Parthenogenetic EmbryoIn this experiment,we established a new chimeric culture pattern of 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.1.1 Study on the activation methods of diploid parthenogenetic embryoAs we knows,parthenogenetic embryo of mammal has several chromosome karyotype.The formation of the different chromosome karyotype is related to the activation methods.The heterozygosis diploid,which has the similar chromosome qualitative to the sexual reproduction embryo.In order to receive these embryos,our reseach use two chemistry methods to active the KM mouse oocyte and inhibit the discharging of the second polar body,observing whether there are two pronucleus formed under the microscope to determine the formation of the heterozygosis diploid. This experiment based on choosing a optimal parthenogenetic activation method to form the heterozygosis diploid which used to form chimera contained 8-cell stage parthenogenetic embryo and male embryo.The result shows:adopting manipulation medium which contain 10mmol/L Src12 and 5ug/mlCB to deal with the oocytes for 4 hours gains the highest activation rate(76.7%).,the formation ratio of the heterozygosis diploid is 39.5%,which is higher than the parthenogenetic embryo ratio activating by alcohol and 6-DMAP,but the difference is not significant(p＞0.05).1.2 Reserch on the cultivate system of the mouse parthenogenetic embryo in the nonageAfter parthenogenetic activation of the mouse oocytes,choosing the heterozygosis diploid embryos,cultured in different culture solution after the parthen-activation,and carry out statistical analysis in order to establish a consummate in vitro culture system of parthenogenetic embryo.the results as follow:(1)CZB and mM16 can sustain 2-cell embryos to develope to blastula.after 24 h cultivation,the 2-cell embryos development rate of different groups is close to each other.but 4-8-cell embryos development rate of CZB+FBS group and CZB+BSA group are higher than mM16 group,these embryos have a faster rate of development.,after 48 h cultivation,no matter CZB or mM16 culture solution which has FBS can get the higher development rate of morula than within BSA,and CZB group with FBS is significant deviation with other groups.(2)using CZB with FBS cultivate 2 cell,after 48 h adding glucose group has a better effect than no adding group,the morula development rate of these two groups is higher than whole range adding group.The result indicate that,when cultivating parthenogenetic embryo in vitro,we should use CZB+15%FBS as culture solution before 4 cell stage,then exchange CZB+15%FBS+Glu to cultivate.1.3 Research on the best time to obtain the sexual reproduction 8-cell stage embryos which are used to sex identificationIn order to receive more 8-cell stage embryos,on the base of the law of mouse development, this experiment spirt the embryo through uterine tube,coition after injecting the HCG for 60,64 and 68h,statistics the developmental stage of the mouse embryos we found that more 8-cell stage embryos can be gained 64h after inject HCG,occupying 76.9%of the whole number of embryos, significant different with 60h after injecting HCG(33.8%,p＜0.05),but has no significant different with 68h after injecting HCG(63.6%,p＞0.05).Some of the embryos have developed to morula 68h after injecting HCG,so we can receive more 8-cell stage embryos 64~68h after HCG.1.4 Research on the gomphosis development of the blastular which contain parthenogenetic embryo and male embryoUsing double PCR sex identification to identify the sexual reproduction 8-cell stage embryo, select the male embryos to gomphosis with the the 8-cell stage parthenogenetic embryo,besides, taking the development of sexual reproduction 8-cell stage embryo and parthenogenetic embryo as control group to observe the development of the chimeric embryo,the purpose is to research the development of the blastular which contain parthenogenetic embryo and male embryo,the results shows that the chimeric embryo can developed to blastula in vitro and the rate of the blastula development is 39.4%,a little more than 35.1%of parthenogenesis 8-cell stage,but there is no significant different(p＞0.05),but has significant different with the rate of blastula development of the sexual reproduction in vitro(90.6%,p＜0.05).2 The clonogenicity of the vicariance cell which derived from the inner cell mass of the parthenogenetic blastula and the rate of the different sex sourseThis research based on the study mode of the KM mouse male embryo gomphosis with parthenogenetic embryo established by double PCR sex identification,further,we study several critical questions,which affect the chimera contains two kinds of embryo cells,as a groundwork,we use double PCR sex identification again to mark the clonogenicity of the vicariance cell which derived from the inner cell mass of the parthenogenetic blastula and the rate of the different sex sourse,establishing the foundation of the remedial parthenogenetic embryo stem cell for further research.2.1 Isolation,culture and preparation of feeder layer of mouse embryonic fibroblastsIn this experiment,we collect 13.5d mouse fetus to separate mouse embryonic fibroblasts.after one day we can see bulk primary generation mouse embryonic fibroblasts are adherence and most of them are fusiform,a small quantity fibroblasts are radial morphology,and also a few round or anoma other cells in them.After 3 d these cells can overgrow bottom of culture flask and show long fusiform.sellecting good third generation MEF to prepare feeder layer on quadripuntal plate which is custodited by gelatin after conducted by mitomycin.2.2 The research on the vicariance cultivation of the blastula inner cell mass derived from the chimera by male embryo and parthenogenetic embryoThis experiment take the single cell isolation from the inner cell mass of six parthenogenetic blastula,it't may collect 11.83 monoplast on average,cultivating and generating for the monoplast in vitro,we found that the monoplast of five parthenogenetic blastula ICM can be cultivated in vitro. Each parthenogenetic blastula may obtain 1.67 cell colony on average.2.3 Study on the rate of different sex source derived from the chimera blastula ICM vicariance cellSex identification to microcolony of the the ESCs obtain from the chimera using the duplexing PCR,the ratio of the parthenogenetic cells and male cells is average 5;12 in the microcolony.The result indicate that the double PCR can detect the source of the parthenogenetic embryo cells from chimera blastula ICM.Conclusion:adopting manipulation medium which contain 10mmol/L Srcl2 and 5ug/mlCB to deal with the oocytes for 4 hours gains the highest heterozygosis diploid parthenogenetic embryo, cultivating in the CZB+15%FBS medium growth to the 4-cell stage and then change the medium with CZB+15%FBS+1.1mg/mLGlu,remove the zona pellucida after developed to the 8-cell.More 8-cell stage embryos can be gained 64～68h after inject HCG by spirting the embryo through uterine tube,removing the zona pellucida and then Sexing by dual primer double PCR.Selecting the 8-cell stage male embryo polymerize with zona pellucida free parthenogenetic 8-cell embryo by WOW,the chimera can develop to chimera in vitro.Straggling the blastula ICM to monoplast,and then cultivate on the MEF can obtain the cell colony.Sex identification to microcolony of the the ESCs obtain from the chimera using the duplexing PCR,the ratio of the parthenogenetic cells and male cells is average 5;12 in the microcolony.The result indicate that the double PCR can detect the source of the parthenogenetic embryo ceils from chimera blastula ICM.