【作者】 吕培茹；

【导师】 卢克焕； 卢晟盛；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本研究主要探讨巴马小型猪体细胞核移植和食蟹猴-猪异种核移植的相关技术问题。本论文包括两大部分,第一部分是文献综述,第二部分是试验研究。试验研究包括:(一)猪和食蟹猴体细胞培养体系的建立;(二)巴马小型猪体细胞核移植体系的建立;(三)PHA对孤雌激活和体细胞核移植胚胎发育的影响;(四)猪体细胞核移植胚胎移植方法的建立;(五)食蟹猴-猪异种核移植的初步研究。试验研究的方法和结果可归纳为如下几点:1.建立巴马小型猪睾丸成纤维细胞、耳部成纤维细胞、普通猪胎儿成纤维细胞和颗粒细胞的体外培养体系,并探讨其作为猪体细胞核移植供体的可能性。使用酶消化和组织块法成功分离培养了睾丸成纤维细胞,鉴定了其细胞类型,并且对其细胞周期与核心组蛋白(H3K9)乙酰化的关系进行了研究。结果表明,该培养体系可以支持上述几种细胞的体外生长。免疫荧光染色鉴定所分离到的细胞为睾丸成纤维细胞。两种同期化方法(血清饥饿和接触抑制)的结果显示:随着血清饥饿时间的延长G0+G1细胞的比例急剧升高后又趋于稳定,2d、4d组与70-80%汇合组差异显著(75.9%,95.9%vs.95.2%,P＜0.05),但2d、4d组差异不显著;细胞随着汇合度的增加G0+G1细胞的比例开始上升,接触抑制两天后基本趋于稳定,2d组、4d和6d组显著高于70-80%汇合组和100%汇合组(97.3%,95.0%,97.4%vs.74.7%,84.2%,P＜0.05)。流式细胞仪分析显示:无论是血清饥饿还是接触抑制处理,G0/G1期供体细胞组蛋白乙酰化水平变化趋势基本相同,基本上是先升高后又降低。2.建立了食蟹猴耳部成纤维细胞的体外培养体系,并探讨其作为猴同种体细胞核移植或异种体细胞核移植供体的可能性。分别使用酶消化和组织块法分离到了食蟹猴耳部成纤维细胞,间接免疫荧光法鉴定了其细胞类型并进行了核型分析。结果表明:该培养体系可以很好地支持该种细胞的体外生长,目前已传代到43代,生长仍旺盛;免疫荧光染色鉴定分离到的细胞为耳部成纤维细胞;对21代的细胞进行核型分析,核型正常。3.本试验的目的是:A.掌握猪卵母细胞成熟过程中第一极体的位置随时间发生偏移的规律,为提高盲吸法去核效率提供依据;B.初步探讨胎儿成纤维细胞和颗粒细胞核移植效率;C.胚胎培养时添加胰岛素对孤雌激活和颗粒核移植胚胎发育率的影响;D.探讨简易融合仪对孤雌激活和体细胞核移植胚胎发育能力的影响。结果表明:在44-46h,有84.7%的极体位置偏移不超过30°,盲吸法的去核率基本与之对应,平均在88.1%,适于在本段时间进行去核。胎儿成纤维细胞、颗粒细胞核移植胚胎的囊胚率分别为9.9%和8.7%。Insulin无论对于颗粒细胞核移植胚胎(8.0%vs.2.4%,P＞0.05),还是孤雌激活胚胎均无显著促进作用(47.3%vs.44.3%,P＞0.05),但孤雌激活胚胎的囊胚显著高于核移植胚胎(47.3%和44.3%vs.8.0%和2.4%,P＜0.05)。简易融合仪下,对于核移植胚胎,场强为200v/mm组的桑椹胚发育率均高于220v/mm组(33.3%,29.6%vs.17.1%,13.8%,P＞0.05);场强一定时,20μs组的桑椹胚率均高于40μs组。随着场强的升高和脉冲时间的延长,桑椹胚发育率呈下降趋势。对于孤雌激活胚胎,20μs组的分裂率和囊胚率均高于40μs组,但差异不显著。;4.对睾丸成纤维细胞进行血清饥饿和接触抑制处理,寻找适宜的同期化方法,同时探讨了高代睾丸成纤维细胞重构胚胎的核重塑的规律,并比较了高代、低代睾丸成纤维细胞和耳部成纤维细胞重构胚胎的发育率。结果显示:接触抑制法处理供体,核移植胚胎融合率显著高于血清饥饿法(68.6%vs.55.3%,P＜0.05),胚胎囊胚率也高于后者(21.1%vs.14.1%;P＞0.05),但差异不显著;重构胚胎激活后3h,供体核的大小基本不发生变化,激活后6h,绝大多数重构胚形成膨大的类原核,到12h时,几乎所有重构胚形成膨大的类原核,并开始观察到重构胚分裂现象;低代睾丸成纤维细胞和耳部成纤维作为供体细胞时,重构胚胎的融合率均显著高于高代睾丸成纤维细胞(84.4%,79.1%vs.69.2%,P＜0.05),前两者的重构胚融合率也差异显著(84.4%vs.79.1%,P＜0.05),低代睾丸成纤维细胞作为供体细胞时重构胚胎的囊胚率显著高于高代睾丸成纤维细胞和耳成纤维细胞(13.9%,8.9%vs.6.4%,P＜0.05)。5.探讨了植物凝集素(PHA)对于化学激活、电激活孤雌激活胚胎和体细胞核移植胚胎发育能力的影响。结果表明:添加适量PHA可以提高化学激活胚胎的囊胚率,10μg/mlPHA可以使囊胚率提高到54.1%;加入量大则有害;添加10μg/ml,20μg/ml的PHA可以显著提高体细胞核移植囊胚发育率(13.0%,5.6%vs.5.0%,P＜0.05),10μg/ml组的囊胚率比对照组提高1倍多。总之,PHA对于化学激活、电激活和体细胞核移植胚胎的作用呈剂量依赖性,适量添加有助于三种胚胎的发育,过量则有害。6.以胎儿成纤维细胞和新生巴马小型猪睾丸成纤维细胞为供体构建的重构胚胎,利用刺入式胚胎移植法将其分别移入两头巴马小型猪和两头陆川猪的输卵管内。其中两头巴马小型猪和一头陆川猪返情,一头陆川猪怀孕到期,产下一头健康的雄性巴马小型猪。说明新生巴马小型猪睾丸成纤维细胞核移植胚胎可以发育到期。7.采用食蟹猴耳部成纤维细胞作为核供体构建猴-猪异种核移植胚胎,研究其核重编机制并寻找适宜的培养基,初步建立食蟹猴-猪异种核移植体系。结果显示:(1)重构胚胎激活后3h,供体核的大小基本不发生变化,但在激活后6h大部分形成膨大的类原核。(2)食蟹猴-猪异种核移植胚胎融合率为74.2%,有70.5%的重构胚胎发生卵裂,29%的卵裂胚胎发育到桑椹胚阶段。(3)NCSU23+10%FCS和TCM199+10%FCS培养基中,重构胚突破4-细胞阻滞发育到8-细胞以上的比例分别为23.5%和17.0%,NCSU23培养基为19.9%,但是只有在NCSU23+10%FCS和TCM199+10%FCS培养基中重构胚胎可以发育到囊胚阶段(1.4%vs.1.1%,P＞0.05),尽管差异不显著。更多还原

【Abstract】 The aims of this study were to investigate issues related to Guangxi bama mini-pig somatic cell nuclear transfer(SCNT)and monkey-pig interspccies somatic cell nuclear transfer.This thesis was arranged with two parts.The first part was dealt with literature review and the second part was with regard to the experimental studies which included:(1)establishment of somatic cell line from Guangxi Bama mini-pig and monkey;(2)establishment of system of Guangxi bama mini-pig somatic cell nuclear transfer;(3)Effect of PHA on parthenogenetic activation and cloned embryo development;(4)establishment of method for transfer of cloned embryos in pig;(5)primary investigation on monkey-pig interspecies somatic cell nuclear transfer.Summaries of this study could be made as the followings.1.Four somatic cell lines were set up from Guangxi Bama mini-pig testicle fibroblast cells and ear fibroblast cells;pig fetal fibroblast cells and granulose cells.It was also investigated whether these cells could be used as donor cells for SCNT.Cells from a new-born Guangxi Bama piglet were isolated and cultured by enzyme-digesting and tissue-piece methods and their cell types were identified by fluorescent immunocytochemistry(ICC).Furthermore,the relationship between cell cycle synchronization and nucleus histone acetylation levels were also investigated.It was concluded that the culture system was good enough for these fibroblast cells.The result of(ICC)indicated that isolated cells from testicle tissues were testicle fibroblast cells.The cells were synchronizated by serum starvation or contact inhibition.With the extending of treatment time in serum starvation group,synchronizated G0/G1 cells inereased rapidly and then kept steady after 2d.Synchronization effiency were sighnificantly higher in treated 2d,4d group than 70-80%conflence group(75.9%,95.9%vs.95.2%, P＜0.05);Similar result was also obtained in contact inhibition group. Synchronization efficiency were sighnificantly higher in 2d,4d and 6d contact inhibition group than the 70-80%and 100%conflence group(97.3%,95.0%, 97.4%vs.74.7%,84.2%,P＜0.05).The level of nucleus histone acetylation in G0/G1 donor cell was similar trend with different treated time by serum starvation or contact inhibition.The level of nucleus histone acetylation increased to the highest level at 2d,then decreased at lowest level.2.Long-tailed macaque ear fibroblast cell was isolated and cultured in vitro and whether it could be used for donor cells in intra-species and inter-species cloning was also investigated.Fibroblast cells were isolated and cultured by enzyme-digesting and tissue-piece methods.Cell type was also identified by fluorescent immunocytochemistry(ICC)and the result indicated that isolated cells were fibroblast cells.The result of chromosome analysis showed that ear fibroblast cells still kept normal when cultured up to passage 21.Now ear fibroblast cells have been passaged 43 times,but still proliferate well.3.The purpose of this experiment was the followings:A.to investigate the moving rule of the first polar body in oocyte maturation so as to improve enucleation efficiency.B.to investigate the development capacity cloned embryos from fetal fibroblast cells and granulose cells.C.to investigate the effect of insulin on development of pathenogenetic and cloned embryos.D. to investigate the development capacity of parthenogenetic and cloned embryos using simple fusion equipment.Results showed that after the oocyte maturation for 44-46h,84.7%polar body moving-angle aren’t beyond 30°.During this time 88.1%oocytes were successfully enucleated by blind-enucleation method;there was no significantly positive effect of insulin on the development of pathenogenetic(47.3%vs.44.3%,P＞0.05)and cloned embryos(8.0%vs.2.4%, P＞0.05);for the simple fusion equipment,the morula rate of cloned embryos was a litter higher in 200v/mm group than 220v/mm group,although there were no significant difference(33.3%,29.6%vs.17.1%,13.8%,P＞0.05);20μs pulse duration group was a little higher than 40μs group when used the same field strength.It indicated that as the field strength and pulse duration increased,the development rate of morual fell down;for pathenogenetic embryos,the blastocyst rate at 20μs group was a little higher than that at 40μs group when 200v/mm field strength was used,but there was no significant difference.4.the optimal cell cycle synchronization protocol of new-born Guangxi Bama mini-pig testicle fibroblast cell(passage3-5)was investigated by contact inhibition and serum starvation;Bama mini-pig testicle fibroblast cell (passage10-14)was introduced into the enucleated oocytes to investigate whether it could be reprogrammed after activation;to determine the potential of the testical fibroblast cells as donor cell,the in vitro developmental capacity of the cloned embryos were examined using low(3-5)and high passage(10-14) testical fibroblast cells,compared to ear fibroblast cells.Result showed that the fusion rate was significantly higher in contact inhibition group than that in starvation group(68.6%vs.55.3%,P＜0.05).The rate of cleaved embryos was similar between the two groups(83.8%vs.82.8%,P＞0.05),while development rate to the blastocyst was higher in contact inhibition group than that in the starvation group(21.1%vs.14.1%,P＞0.05);At 3 hours after activation,the nucleus was clearly visible and with the same size as the donor cell. Enlargement of the nucleus was investigated at 6h after activation.At 12h after activation the number of the reconstructed embryos with enlargement nucleus increased,and some of them even cleaved;there was significant difference in fusion rate using either ear fibroblasts or low(3-5)and high passage(10-14) testicular fibroblasts(84.4%,79.1%vs.69.2%,P＜0.05).Similar cleavage rates were found in all three groups.Low passage testicular fibroblasts resulted in the highest rate of blastocyst production but there was no significant difference between high passage testicular fibroblasts and ear fibroblasts.5.The effect of PHA in culture medium on parthenogenetic and cloned embryos development was examined.Result showed that parthenogenetic embryo development to the blastocyst stage in the presence of PHA(10μg/ml)was higher than in control group(0μg/ml),but there was no significant difference; however,the percentage of cloned embryo development developing to the blastocyst stage in the presence of PHA(10μg/ml)was significantly higher than that with treatment group(20μg/ml)and control group(0μg/ml)(13.0%vs.5.6%, 5.0%,P＜0.05).In a word,the influence of PHA on parthenogenetic and cloned embryo was dose-independent.Treatment of 10μg/ml PHA is the optimal concentration to sustain cloned and parthenogenetic embryos development.6.In order to evaluate the development capacity of cloned embryo in vivo,the reconstructed embryos from fetal fibroblast cells and bama mini-pig testicle fibroblast cells were penetrated into the oviducts of the surrogate gilts with a needle.A total of 4 surrogate gilts were used and two of them were bama min-pig and the other two were Guangxi Luchuan pig.Result showed that both two Bama mini-pig surrogate returned to estrus.One of the two luchuan gilts gave birth to one healthy male Bama mini-pig.The cloned embryos using Bama mini-pig testicle fibroblast cells as donor cell could develope to term in vivo.7.Long-tailed macaque ear fibroblast cells,as donor cells,were introduced into pig enucleated oocytes to investigate whether they could be reprogrammed. To investigate appropriate embryo culture conditions for interspecies cloned embryos,three culture mediums were used:NCSU-23,NCSU-23+10%FCS, TCM199+10%FCS.Results showed that 6h after activation,the introduced nucleus gradually became swollen,which indicated that monkey ear fibroblast cells could be reprogrammed.The fusion rate of monkey-pig reconstruct embryos was 74.2%;70.5%of reconstructed embryos cleaved and 29%cleaved embryos developed to morula;all the three culture media could help the reconstructed embryos break through 4-cells block and develop to beyond 8-cells(23.5%,17.0%vs 19.9%,P＞0.05).And a few reconstructed embryos developed to blastoeysts(1.4%vs.1.1%,P＞0.05)in media of NCSU23+10%FCS,and TCM199+10%FCS,but there was no significant difference between these two media.更多还原