【作者】 李向臣；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2007， 博士

【摘要】 实验一:卵母细胞的成熟在核移植的过程中有很重要的作用。本实验比较了:①不同直径大小的牛卵泡液对牛卵母细胞体外成熟的影响。添加10% FBS成熟液作为对照组,实验组为直径>8 mm 10%牛卵泡液(BFF)、2～8 mm 10% BFF及5% >8 mm BFF+2～8 mm 5% BFF,成熟18h去掉卵母细胞的颗粒细胞,比较卵母细胞成熟率及激活处理后的囊胚发育率。结果表明:对照组与实验组之间成熟率无显著的差异(P>0.05),>8 mm 10% BFF囊胚率(43.40%)显著的高于对照组,添加卵泡液的混合组囊胚率也显著高于对照组,说明卵泡液的添加能有效提高牛卵母细胞孤雌囊胚发育率。②卵泡液对重组胚发育的影响。实验①研究发现,大卵泡中粘多糖的存在不利于卵母细胞进行重组胚的操作,因此减少了>8mm的BFF;以10% FBS为对照组,实验组为5% BFF(2～8 mm)+ 5% FBS、5% (>8 mm 2.5% +2～8 mm 2.5%,混合)+ 5%FBS及10% BFF(2～8 mm),成熟18h后选择成熟卵母细胞去核构建重组胚。5%(>8 mm 2.5% + 2～8 mm 2.5%,混合)+ 5% FBS囊胚率(26.56%)与其它组比差异显著。卵泡液较FBS能提高牛卵母细胞孤雌胚和核移植胚的发育。实验二:第一次利用免疫组织化学的方法对牛卵母细胞成熟过程β-tubulin的变化过程进行了初步的研究。结果利用Nikon公司TE 300荧光显微镜发现,0、6h不能见到β-tubulin的分布, 12h有微弱的荧光,14hβ-tubulin围绕在凝集的染色质周围,荧光相对较弱,16h荧光较强,说明牛卵母细胞在14～16h生发泡破裂排出第一极体。作为阴性对照,卵母细胞未能见到荧光。实验三:去核是核移植过程中关键重要的一步,实验利用蔗糖和脱羰秋水酰碱(Dmemcolcine,DM)进行了显示去核和诱导去核的研究。①在核移植的操作液中添加0、1%、3%、5%、7%、10%(v/v)蔗糖孵育1h,仅在3%组中发现74枚卵母细胞有3枚显示;②将牛成熟卵母细胞用离子霉素(Ion)激活5 min,于添加0、0.4、0.5、0.6μg.mL-1DM的SOFaa培养液中孵育1.5 h诱导去核,未发现诱导去核的卵母细胞。③牛卵母细胞激活5min后在0.5μg.mL-1 DM中孵育0.5、1、1.5、2h诱导去核同样未见到能完全诱导去核的现象;④将成熟牛卵母细胞置于0、0.4、0.5、0.6μg.mL-1 DM中孵育2h,加入DM之后发现牛成熟卵母细胞染色体以一种类似“芽孢”样的方式凸出于卵母细胞的胞膜上,0.5μg.mL-1 DM显著高于其它组(P <0.05),而在对照组中未出现显示核的现象;⑤将牛成熟卵母细胞在DM中孵育0.5h、1h、2h,以孵育2h的显示效率最高(76.54%),与0.5h、1h相比差异显著(P <0.05);⑥实验比较成熟不同时间卵母细胞对显示去核的影响,发现在添加0.5μg.mL-1DM孵育2h中,成熟18 h极显著的高于14h和16h组(P<0.01),与20h成熟组的相比没有显著的差异;⑦实验对成熟的过程中添加DM进行了研究,添加DM孵育2h后再去颗粒细胞,研究表明在成熟18h添加DM既能提高其显示去核的效率同时与其他组相比也对囊胚发育率有所提高,18h、16h组与14h组相比差异显著。结果表明:本实验利用蔗糖不能达到理想的显示核的效果,同时DM也未能起到诱导去核的目的;首次发现利用DM可以起到显示核的作用。实验四:本实验研究了Ion+6-DMAP、乙醇+6-DMAP对重组胚的激活。结果表明:在Ion+6-DMAP组中,Ion激活5min卵裂率最高,与激活3min、7min和10min相比有显著的差异(p﹤0.05),且囊胚率也显著高于其它组(p﹤0.05);乙醇+6-DMAP组中,以激活5min效果比较明显,卵裂率与3min、7min和10min比有显著的差异(p﹤0.05),卵裂率达56.53%,囊胚率在8.51%,说明两组激活方式均能激活牛的重组胚,但是相比以Ion+6-DMAP组激活较好。实验五:实验分析供体细胞对重组胚发育的影响。①0.5%血清饥饿0d、1～3d、4～6d、7～9d四组实验中,重组胚卵裂率没有显著的影响(P>O.05),囊胚以饥饿1～3d效果较好;②细胞传代1～3代用于核移植,桑椹胚达到32.06%的效果显著高于0、4～6代、7～9代组(p﹤0.05);③冷冻的细胞解冻之后培养24h,饥饿1～3d,发现解冻2、4、6代的细胞重组胚的桑椹胚率差异不显著,较8代解冻的细胞用于核供体效果好(p﹤0.05)。实验六:本研究对重组胚进行了微卫星DNA多态性的分析。结果表明,体细胞克隆胚胎和核供体细胞的11个STR位点的PAGE基因型完全一致。更多还原

【Abstract】 Experment 1.It is important the nuclear transfer process that oocyte mature.This experiment was desgined:①w e added different diameter size bovine liquor folliculi(BFF)to mature fluid in vitro.10% FBS mature fluid was for control group.Our test contained the diameter > 8 mm 10% cow liquor folliculi (BFF), 2～8 mm 10% BFF and 5% > 8 mm BFF + 2～8 mm 5% BFF.After mature 18h removes the ovocyte the granular cell and compared t mature rate and activated oocytes the rate of blastocyst rate.The result indicated that, Between the control group and the experimental group does not have the remarkable difference (P>0.05)in the maturing rate.Blastocyst rate(43.40%)with > 8 mm 10% BFF is remarkable higher than control group. Mix group blastocyst rate is also remarkably higher than control group.So BFF can effectively enhance the development rate of bovine parthenogentic blastocyst.②Effect of BFF on reconstructed embryos.Tests①research to discover.Because mucoitin with big BFF does not favor to carry on the reconstructed embryos operation.Therefore reduced > 8mm BFF. Take 10% FBS as control group, the experimental group is 5% BFF (2～8 mm) + 5% FBS, 5% (> 8 mm 2.5% +2～8 mm 2.5%,Mix) + 5%FBS and 10% BFF (2～8 mm) respectively.We used matured oocytes to reconstruct embryos after mature 18h. 5% (> 8 mm 2.5% + 2～8 mm 2.5%,The mix) + 5% FBS pouch embryo rate (26.56%) and other groups are more remarkable than the difference. The development rate of reconstructed embryos in 5% (>8 mm 2.5% + 2～8 mm 2.5%,mix) group and 5% FBS group is higher significantly than others group is treated (p<0.05).The BFF compares FBS to be able to enhance The development of bovine parthenogentic blastocyst and reconstructed embryos.Experment 2.First time has conducted the preliminary research using the immunity histochemistry method to the bovine oocytes mature processβ-tubulin change process. The result discovered using Nikon Corporation TE 300,oocytes matured for0, 6h cannot see beta theβ-tubulin distribution, 12h has the weak fluorescence, 14hβ-tubulin revolves around the chromatin, fluorescence relative weak, the 16h fluorescence is strong, explained the bovine ovocyte bursts in 14～16h GVBD and discharges the first polarbody. As the negative comparison, the ovocyte has not been able to see the fluorescence.Experment 3.Enucleation of the oocyte is one of the key steps in NT.We researched whether sucrose and demecolcine (DM) treatment is applicable.①Working concentrations of sucrose 0-10%s (v/v) the sucrose incubated 1h.Three oocytes was revealed in seventy-four only with 3% group.②Matured oocytes were activated 5 min with ionomycin (Ion). Adding 0, 0.4, 0.5, 0.6μg.mL-1DM in the media SOFaa in 1.5 h. The oocytes were induced enucleation by DM.③Activated with Ion for 5 min and incubated 0.5、1、1.5、2h respectively to induce enucleation .The same we didn’t find induced enucleation by DM the phenomenon.④Matured oocytes were put in DM with 0.4, 0.5, 0.6 mg.mL-1 for 2h.we found that the majority of the treated bovine oocytes formed a small transparent bud into the perivitelline space.Media with 0.5μg.mL-1 is remarkably higher than other two groups (P <0.05), but has not revealed nucleus in control group.⑤Put matured oocytes in DM for incubated 0.5h, 1h, 2h respectively. It is the highest with 2h (76.54%)than 0.5h and 1h group(P <0.05).⑥The revealed enucleation by different matured time of bovine oocytes were investigated.Adding 0.5μg.mL-1DM in the media incubated 2h and 18h matured oocyted is remarkable higher than 14h and 16h group (P <0.01), compareed not the remarkable difference with the 20h mature group.⑦Adding DM in matured culture media during the mature process was investigated. Granular cell was removed 2h later. We found that increased DM in mature 18h was able to enhance it to revealed enucleation efficiency compared with other groups and enhance the development rate of bovine blastocyst.The development rate of bovine fibroblast reconstructed embryos in 18h group and 16h group is higher significantly than 14h group.The result indicated that using the sucrose cannot achieve the ideal revealed nucleus the effect. At the same time DMcan not induces enucleation the goal. The first time we found DM can be used to revealed enucleation.Experment 4.The bovine reconstructed embryos activated by Ion + 6-DMAP and ethyl alcohol + 6-DMAP procedures were investigated. The result indicated that in Ion + 6-DMAP group, the cleavage rate of oocytes in ion activated for 5min was significantly higher than those in other treatments and the rate of blastocyst was significantly higher than others groups(p﹤0.05); In the ethyl alcohol + 6-DMAP group, reconstructed embryos by activated in the 5min is obvious, 5min group compared with 3min, 7min and 10min,the cleavage rate of 5min group is remarkable higher than others groups(p﹤0.05). the cleavage rate reaches 56.53%, the rate of blastocyst is 8.51%. These results indicate that bovine reconstructed embryos can be activated effectively by Ion + 6-DMAP and ethyl alcohol + 6-DMAP, but ion + the 6-DMAP group is better than ethyl alcohol + 6-DMAP.Experment 5.Effect of the donor cell on development of reconstructed embryos.①Donor cell by serum starvation with 0.5% is hungry 0d, 1～3d, 4～6d, 7～9d respectively. the cleavage of ratereconstructed embryos is not differentnot (P>O. 05).The rate of blastocyst is better in 1～3d group.②Cells passaged for 1-3 generations were used for nucear transfer, morula rate is 32.06%,significantly higher than that of 0,4-6,7-9 generation groups(p﹤0.05).③a fter thawing,cells were incubated for 24h,starvated for 1-3d, morula rate of reconstructed embryo has no difference among 2,4 and 6 generation after thawing,but nucear transfer effect is better than 8 generation after thawing (p﹤0.05).Experment 6.To identify the reconstructed embryos by miemsatellite DNA.By sequence amplification of microsatellite DNA,the reconstructed embryos is completely consistent donor cell 11 STR the position spot PAGE genotype.更多还原