【作者】 付博；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 基因组定点修饰的转基因克隆猪有益于农业生产和人类疾病治疗。尽管,人们通过体细胞核移植技术已经得到了克隆猪和转基因克隆猪,并从中摸索出了一些提高体细胞克隆效率的新方法；然而,由于胚胎体外发育环境的缺陷,体细胞克隆的总体效率仍然较低。本研究旨在建立一套高效的体细胞克隆猪的生产体系。研究了激素、猪卵泡液及表皮生长因子对猪卵母细胞体外成熟的影响。系统地探讨了供核细胞、非洲绿猴肾细胞共培养体系及培养基中能量底物变化对猪核移植胚发育的影响。同时,本研究探讨了曲古抑菌素A(组蛋白去乙酰化抑制剂)对转基因细胞中EGFP的表达及转基因核移植胚发育的影响,为转基因猪的生产奠定基础。激素、猪卵泡液、表皮生长因子对猪卵母细胞成熟有促进作用。卵母细胞体外成熟培养过程中,添加10 IU/ml PMSG,10IU/mlhCG至培养液中,培养22h后撤除激素,卵母细胞的成熟率显著高于其他处理(P<0.05)。当培养液中添加10%的猪卵泡液时,卵母细胞的成熟率显著高于其他处理(P<0.05)。当培养液中添加表皮生长因子时,10ng/ml处理组的卵母细胞成熟率显著高于0ng/ml和5ng/ml处理组；然而,10ng/ml处理组和15ng/ml处理组的卵母细胞成熟率无显著差异(P>0.05)。由于大白猪胎儿成纤维细胞易分离、生长快、传代能力强,所以选用该细胞作为核供体细胞。对不同传代次数的细胞进行染色体分析。结果表明,传代7次细胞的正常核型比例在80%左右,满足核移植的要求。随着供核细胞传代次数的增加,重构胚的囊胚率下降。用表面光滑细胞及表面粗糙细胞分别作为核移植供体,表面光滑的细胞可以提高供核细胞与受体卵母细胞的融合率。Yasumura等人从成年非洲绿猴的肾脏分离得到Vero细胞系。尽管大量的文献都表明Vero细胞对哺乳动物的胚胎发育有促进作用,然而,Vero细胞对猪胚胎发育的影响尚未见报道。本文首次使用Vero细胞作为滋养细胞,旨在探讨共培养体系中Vero细胞对猪孤雌胚及核移植胚发育的影响。结果显示,Vero细胞共培养组的孤雌胚囊胚率及核移植胚囊胚率显著高于对照组(P<0.05),即Vero细胞共培养体系对猪孤雌胚及核移植胚的发育都具有促进作用。将NCSU-23培养基中的5.56 mmol/L葡萄糖替换为0.2 mmol/L丙酮酸、5.7 mmol/L乳酸,并将此培养基命名为mNCSU-23。根据实验设计,孤雌胚及核移植胚转移到mNCSU-23或NCSU-23中培养。激活第2天统计孤雌胚及核移植胚中的5-8细胞胚胎数。激活第6天统计孤雌胚及核移植胚囊胚形成率及囊胚细胞数。实验结果表明,mNCSU/NCSU处理组的囊胚率显著高于对照组(P<0.05)；单纯使用mNCSU-23培养猪胚胎时,囊胚率最低,发育结果最差(P<0.05)。结果证实,在体外培养前两天,用乳酸和丙酮酸代替培养基中的葡萄糖对猪胚胎发育有利。目前,外源基因导入真核细胞的方法主要包括：病毒感染法、原核注射法、DEAE-葡聚糖转染法、电穿孔法、脂质体转染法等。脂质体法操作简单,不需要昂贵的仪器设备,可大批量转染,且对细胞毒性作用小。故本实验采用脂质体法将pEGFP-C1载体导入大白猪胎儿成纤维细胞中,并对转染参数进行了优化：脂质体4μl,质粒DNA 2μg,稳定转染6小时效率较高。将pEGFP-C1质粒载体转染入大白猪胎儿成纤维细胞。经G418筛选后,得到稳定转染的阳性细胞。随传代次数增加,EGFP阳性细胞的比例下降。用曲古抑菌素A预处理转基因供核细胞,同时设立对照组(供核细胞不使用曲古抑菌素A预处理)。蓝光照射转基因供核细胞和转基因的重构胚,观察供核细胞和转基因重构胚中EGFP的表达情况。结果表明,当用50nM曲古抑菌素A处理供核细胞时,供核细胞的EGFP阳性率显著高于对照组；当用50nM曲古抑菌素A处理供核细胞时,重构胚中EGFP的阳性胚率显著高于对照组(重构胚融合激活48h后),且这种EGFP表达的增加至少维持到桑椹胚阶段。此外,TSA处理对转基因细胞有毒性影响,且这种毒性影响是剂量依赖的；当曲古抑菌素A浓度高于50nm时,大部分细胞死亡。用50nM曲古抑菌素A处理供核细胞后,细胞形态发生变化,即细胞拉长、细胞轮廓不清晰。更多还原

【Abstract】 The production of transgenic cloned pigs with site specific alterations would have great benefits in agriculture as well as human therapeutic applications. Although dozens of nontransgenic and transgenic pigs have been born after somatic cell nuclear transfer, and several new methods have been introduced, the overall efficiency remains low due to the poor in vitro embryo development. This research aim to establish an efficient system for producing pigs by somatic cell nuclear transfer(SCNT).Effect of hormone、pFF and EGF on maturation of porcine oocytes have been investigated. With regard to the development of porcine nuclear transfer embryos, systematical study had been carried on factors including donor cells、co-cultural systems with Vero cells and energy substrate. At the same time, this study pertain to investigating the effect of TSA(a histone-deacetylase inhibitor) on EGFP expression in transfected cells and embryonic development after SCNT, providing theoretical significance for producing transgenic pigs.Hormone、PFF and EGF have positive effects on maturation of porcine oocytes. When treated with 10 IU/ml PMSG,10 IU/ml hCG for first 24h,oocytes have higher rate of maturation than other treatments(P<0.05). When pFF was added into maturation medium, rate of maturation in 10%pFF treatment was significantly higher than other treatments(P<0.05). When oocytes were treated with EGF, the rate of maturation was significantly higher in 10ng/ml group than that in 0 ng/ml and 5ng/ml groups(P< 0.05); there was no significant difference between lOng/ml group and 15ng/ml group(P>0.05).Yorkshire fetal fibroblasts can be used as nucleus donator in somatic cell nuclear transfer due to the characteristic of easy to be separated and rapid growth velocity, strong passage capability, more amplification generations in vitro culture. Chromosome analysis was performed in different cell passages. Normal rate of chromosome nuclear type can reach to 80% when cultured up to passage 7, which satisfied the requirement of somatic cell nuclear transfer. Smooth cells or rough surface cells were used as donor in SCNT. Smooth surface cells led to a higher fusion rate. With the number of passage of donor cells increasing, the rate of blastocysts formation decrease.The Vero cells line was derived from the kidney of a normal, adult, African green monkey on March 27,1962, by Y. Yasumura and Y. Kawakita at the Chiba University in Japan. Despite the abundance of literature on the positive effects of Vero cells on some mammalian embryonic development, there are few reports about co-culture system with Vero cells throughout the culture interval of porcine embryos.This experiment was conducted to study the effect of Vero cells in embryo co-culture system on the efficiency of blastocyst production in pig. The result showed that groups co-cultrued with Vero cells achieved significantly higher blastocyst formation rate compared with control(P<0.05).Co-culture with Vero cells improve the development of porcine embryos in vitro. This is the first time that Vero cells were used as feeders in producing cloned pigs.This study also investigate the effects of pyruvate and lactic acid on the earlier development of porcine embryos.5.56 mmol/L glucose in culture medium (NCSU-23) was replaced with 0.2 mmol/L pyruvate and 5.7 mmol/L lactic acid, namely mNCSU-23. Parthenogenetic embryos and nuclear transfer embryos were transferred into NCSU-23 or mNCSU-23 medium according to the experimental design. Parthenogenetic embryos and nuclear transfer embryos were evaluated for the numbers of 5-8 cells stage on Day 2. Blastocyst rates and the numbers of nuclei in the blastocyst were determined on Day 6. We observed that the rates of blastocysts formation in mNCSU/NCSU treatments on Day 6 were significantly higher than control, with mNCSU-23/mNCSU-23 treatment having the lowest rates of blastocysts formation on Day 6 (P<0.05). Our results have demonstrated that replacing glucose with pyruvate and lactic acid during the first part of IVC may be beneficial to the development of the porcine embryo.At present, there are many methods for exogenous gene to introduce eukaryotic cell, such as Viral infection, Pronuclears microinjection, DEAE-glucosan infection protocol, Electroporation, Lipofectamin-mediated DNA transfection method and so on.Liposome infection protocol is simple, doesn’t needs expensive instrument, can transfection a great deal of cells and less harm to cell. So in this study, pEGFP-Cl vector was introduced into fetal fibroblast of Yorkshire by lipofectamin-mediated DNA transfection methods, transfection parameters were optimized. Lipofectamin 4μl, plasmid vector DNA 2μg, stable transfection 6 hours are optimized condition for transfection. Yorkshire fetal fibroblasts were transfected with pEGFP-Cl plasmid. After selecting with G418, stable transfected cell line was gotten. With the increase of passage number, EGFP expressed cells significantly decreased. Then transfected cells, donor cells for SCNT, were pre-treated with TSA, with the untreated cells used as control.Expression of EGFP in donor cells and re-constructed embryos was detected when exposed to blue light. Results have shown that percentage of EGFP-positive cells significantly increased when the transfected cells were treated with 50nM TSA and percentage of EGFP embryos on day 2 from 50 nM TSA treatment was significantly higher than control groups and increaed transgene expression continues to at least the morula stage. In addition, it appeared that the cytotoxic effect of TSA on the transfected cells was dose-dependent. Most of the cells died when TSA consentation was over 50nM(P<0.05). The cells changed morphologically at 24 h after TSA treatment. TSA-treated cells elongated in size and were vague in outlines.更多还原