牛gv期卵母细胞玻璃化冷冻技术优化及其作用机制研究

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1、牛GV期卵母细胞玻璃化冷冻技术优化及其作用机制研究组(3．2±0．1％)低于mPBS+0．5M海藻糖组(3．9+0．9％)，三组在统计学上差异不显著(矿O．05)。实验结论：DPBS中的钠离子对牛GV期卵母细胞冷冻有一定的危害，以氯化胆碱替代，能起到一定的保护作用，并且非渗透性保护剂0．5mol／L海藻糖替代蔗糖，与氯化胆碱配合使用，能达到更优效果。关键词：牛；GV期卵母细胞；玻璃化冷冻；海藻糖；氯化胆碱华中农业大学2013届硕士研究生学位(毕业)论文AbstractComparedwiththematureoocytecryopreservation，cattleGVoocytecr

2、yopreservationisverydifficult．Butinthelong—distancetransportationofoocyte，cryopreservationisneeded．So，thedevelopmentofGVoocytecryopreservationhavesignificantroleonmakingfulluseofimmatureoocytes，expeciallylong-distancecryopreservationofoocyte．Trehalose，anon-reducingdisaccharide，canformaglassyprot

3、ectinglayerforthealivecellunderextremeconditions．Trehalosecanbeappliedinembryoandoocytecryopreservationasanon-permeablecryoprotectantsinsteadofthesucroseinCPAinthisstudy；somepreviousstudieshadshowedthattheNaCI，asthemainingredientintheconventionalcryoprotectantsbufferDPBSmightcausesomecertaindama

4、geintheembryocryopreservation．Therefore，cholinechloridecouldbeapropersubstituteofNaCIinalleviatingthepotentialdamageofthesodiumions．Inthisstudy,theoptimalsubstitutionproportionforthetrehaloseandcholinechloridetothesucroseandNaCIrespectivelywerefoundthroughcomparingwiththedevelopmentpotential，sur

5、viverateoftheoocytes，celldamageandexpressionsofapoptosisandcyclegenesamongdifferentcryopreservationgroups．Theexperimentalresultswerelistedasfollows：1．ToexplorefrozeneffectofdifferenttrehaloseconcentrationsonGV-stageoocytesvitrification(1)AddeddifferentconcentrationsoftrehaloseintheCPAtodetectthe

6、survivalrates．TheFDAfluorescenceintensityofthefrozengroupO．5MTrehalosewaslessthanthe0．5Msucrosegroup(39．8％VS52．5％，P<0．05)，buttheFluorescencevalueoftheO．5MsucrosegroupWaslessthanthe0．5MTrehalosegroup(63．6％VS70．1％，P<0．05)，Therewasnosignificantdifferencebetweentheotherfrozengroups．(2)Thezonapelluci

7、dadissolutiontimeofallfrozengroupscomparedwiththefleshgroupweresignificantlyincreasedp<0．05)；Thezonapellucidadissolutiontimeof0．5Msucrosegroup(93-I-2．5min)wassignificantlylessthantheotherfrozengroup，Exceptingfor0．5MTrehalose