The Research On Partheno-activation Method To Improve The Formation Rate Of Heterozygous Diploid Blastula

Parthenogenetic stem cell,a new kind of therapeutic cloning embryonic stem cell, owing to its identical MHC with original animal,can apply to histiocytic substitute therapeutics compared to somatic cloning embryonic stem cell.The low rate of cloning blastula development and quality problems of obtained embryonic stem cell,make the applications of somatic cloning embryonic stem cell have some limits.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.At present,the isolation rate of parthenogenetic stem cell from partheno-blastula is very low.The research indicates that the low isolation rate has great relationship with the karyotype of partheno-blastula,while that again has correlation with the karyotype of activated oocyte.Now most researchers apply unknown karyotype blastula to isolate the stem cell without choice.In fact,only blastula cell with heterozygous diploid karyotype has similar development abilities with normal somatic cell in theories.So the karyotype of partheno-blastula maybe influences its development and isolation rate of stem cell seriously.And the different karyotypes of partheno-blastula come from matured oocytes activated by different activators.So the partheno-activation methods have direct relationship with the formation of partheno-blastula karyotypes.In this experiment,oocytes were recovered from mice killed after 18 hours, denuded of cumulus with hyaluronidase.The formation of different parthenogenetic activation type and blastocyst development rate of the resulting parthenogenetic embryos treated with five activators were investigated and by choosing best activation method to produce blastula with heterozygous diploid karyotype to isolate the stem cell in order to find a effective blastula formation method used to isolate the stem cell for cloning therapeutic objectives.Experiment summarized as follows: Experiment 1:The research on partheno-activation methods to improve the rate of blastula with heterozygous diploid karyotype.Oocytes were denuded of cumulus with hyaluronidase and then activated by five activators and then we observe whether Pb₂ discharged or not and process karyotype analysis to investigate the formation of different parthenogenetic activation type treated with five activators and then culture in vitro to develop toblastulas to investigate blastocyst development rate of the resulting parthenogenetic embryos,Which in order to choose the best activation method to produce blastula with heterozygous diploid karyotype.Results:(1)Mouse oocytes could be activated by different treantments and four types of parthenogenetic activation(uniform haploid,mosaic haploid,heterozygous diploid with one pronucleus and heterozygous diploid with two pronuclei)were produced.Alcohol and SrCl₂ alone produced higher rate of uniform haploid,while alcohol united with 6-DMAP and SrCl₂ united with CB produced higher rate of diploid.(2) The activation rate oocytes was 63.3%after treatment with 10%alcohol alone for 10 minutes,and produced higher rate of mosaic haploid,but lower rate of the cleavage rate and blastocyst development rate(6.7%).(3) 10%alcohol treated 5 minutes first and then 6-DMAP for 2,4,6h,the activation rate improved as time(37.8%,39.7%,75.0%).The cleavage rate of oocytes treated with 10%alcohol and 6-DMAP for 6 hours was significantly higher than that for 2 and 4 hours,producing higher rate of 2PN heterozygous diploid and rate of blastocyst development which was significantly higher than 10%alcohol treated alone(16.7%vs 6.7%,P＜0.05).(4) 10mM SrCl₂ alone and united with CB can both activate oocytes efficiently after treatment for 2 and 4 hours.SrCl₂ activation alone produced higher rate of haploid,while higher rate of diploid when united with CB,and cleavage and blastocyst development rates were significantly different (22.7%vs8.1%,P＜0.05).The conclusion:10mM SrCl₂ and 5ug/mlCB treated 4h is the best method to obtain the higher rate of heterozygous diploid blastula.Experiment 2:The effect observation of stem cell isolation with the whole embryo and immune serum methods.The aim is to establish more reliable research flat for stem cell isolation according to existing research,which in order to establish basis for parthenogenetic stem cell isolation.In this experiment,we collect blastula after pregnancy for 3.5 days,then isolate ICM with the whole embryo and immune serum methods to inoculate on feed layer to isolate ESCs colony.Results:(1)We collect 80 blastula,after inoculating on feed layer for 48 hours,72 of which hatch out to stick on feed layer.And after 96 hours,ICM start to proliferate and seek out to disperse to 2～3 cells after 5 days.At last,we obtained 20 ESCs colonies.(2) We collect 78 blastula and get rid of Zona pellucida.ICM are obtained with immune serum method and we got 22 ESCs colonies.(3) Obtained ES clony has typical ESCs shape and AKP check positively.Experiment 3:The primary research on isolation rate of parthenogenetic stem cell from partheno-blastula with uniform haploid and heterozygous diploid karyotype. Accordinf to the conclusion of experiment 1,we obtained heterozygous diploid and uniform haploid partheno-blastula.Considering it's not easy for parthenogenetic blastula to hatch out from Zona pellucida because of long time operation in vitro,we choose immune serum method to isolate the stem cell.we compare isolation rate of parthenogenetic stem cell from two kinds of partheno-blastula in order to choose the type of partheno-blastula with higher stem cell isolation rate.Results:(1)316 oocytes were activated to obtain 68 partheno-blastula with heterozygous diploid karyotype. Only 2 ES clonies were obtained by immune serum method(isolation rate 3%(2/68)).(2)398 oocytes were activated to obtain 68 partheno-blastula with uniform haploid karyotype.Only 1 ES clony was obtained by immune serum method(isolation rate1.7%(1/58).(3)Obtained ES clony has typical ESCs shape and AKP check positively.The conclusion:Heterozygous diploid blastula have higher ESC colony isolation rate than uniform haploid blastula.Conclusions:10mM SrCl₂ and 5ug/mlCB treated 4h is the best method to obtain the higher rate of heterozygous diploid blastula and higher isolation rate of stem cell.