【作者】 张艳丽；

【导师】 王锋；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 人溶酶体β-葡萄糖苷酶(human lysosomal acidβ-glucosidase, GlcCerase)是糖蛋白降解途径的主要外糖苷酶,参与糖蛋白的回收利用。该酶减少或缺失会导致葡萄糖脑苷脂不能有效降解,在各器官中大量沉积,从而使机体发生广泛的病理变化,临床上称为“戈谢病”,酶替代法是目前该病的主要疗法。人体来源的GlcCerase获得极为困难,应用哺乳动物细胞和转基因植物表达系统生产重组人GlcCerase虽然已经有一些成功的报道,但也面临许多难以克服的问题,如在CHO细胞表达重组人GlcCerase,生产成本过于昂贵；在转基因植物中表达,存在重组蛋白质中糖链结构的改变以及下游加工处理困难等限制。如果应用转基因动物乳腺生物反应器生产重组人GlcCerase,在得到天然活性较高产品的同时,将极大地降低生产成本,具有其它表达系统所不能代替的优势。然而,显微注射法的高成本、低效率长期以来制约着转基因动物研究的发展,体细胞转基因与核移植相结合制备动物乳腺生物反应器是当今转基因整合表达的一种有效途径。因此,本研究选取人溶酶体β-葡萄糖苷酶作为研究对象,首先克隆人GlcCerase cDNA序列并对其在COS7细胞中的表达进行初步研究,然后构建含有人GlcCerase cDNA的乳腺表达载体,经体外培养的乳腺上皮细胞验证载体的有效性后,进一步将该载体转染奶山羊胎儿成纤维细胞,筛选稳定转基因细胞克隆株,通过体细胞核移植法生产转基因奶山羊克隆胚胎,以期获得乳腺特异性表达GlcCerase的转基因奶山羊乳腺生物反应器。本研究共分为六个部分,第一、二部分进行了人GlcCerase基因的克隆、表达载体的构建及体外细胞表达研究；第三、四部分主要是分离培养了奶山羊胎儿成纤维细胞,并优化了脂质体法转染该细胞的体系,获得了稳定整合人GlcCerase基因的供体细胞；第五部分利用转人GlcCerase基因的奶山羊胎儿成纤维细胞进行了核移植,研究转基因供体细胞对克隆胚胎体外发育的支持作用,并对克隆胚胎进行了胚胎移植；第六部分探讨了外源基因导入对奶山羊体细胞周期分布、细胞凋亡和基因表达水平的影响。主要的研究结果如下：第一部分人GlcCerase cDNA序列的克隆及真核细胞表达试验中,首先从人胎盘组织中分离提取得到总RNA,利用RT-PCR方法,直接获得人GlcCerase基因的编码序列,经测序分析,所得GlcCerase cDNA与GeneBank中同源性为99%,发现1个碱基差异,导致天冬氨酸到甘氨酸的改变。为了尽快检测所获得的目标基因是否能正常编码获得重组蛋白质,本试验以pEGFP-Cl为基础质粒,构建了含有人GlcCerase基因的真核表达载体pEGFP-GlcCerase。用脂质体介导法将该载体转染入COS7细胞中进行暂态表达研究,可见报告基因GFP顺利表达,经RT-PCR和荧光酶学法进行验证,在细胞中检测到了GlcCerase的mRNA表达,并在细胞裂解产物中检测到了GlcCerase的生物活性。这些结果表明所克隆的人GlcCerase cDNA能够正确编码蛋白,发挥生物学功能,可以用于下一步的乳腺表达载体构建。第二部分人GlcCerase基因乳腺特异性表达载体的构建及乳腺细胞表达试验中,将在体外经真核细胞表达验证正确的GlcCerase基因以及细胞筛选标记基因(Neor)插入含有山羊p-酪蛋白基因调控序列的pBC1载体中,经PCR和酶切鉴定,得到正确的重组质粒pBCl-GlcCerase-Neo。为了检测乳腺表达载体的有效性,将该载体转染入小鼠乳腺上皮细胞系——HC-11细胞中,经G418抗性筛选,获得阳性克隆细胞,将克隆细胞扩大培养后,经PCR检测结果表明,人GlcCerase基因己成功转入到HC-11细胞中。进一步用催乳素、胰岛素及氢化可的松诱导培养转基因细胞,经RT-PCR和Western-blot检测表明,山羊p-酪蛋白基因启动子驱动的人GlcCerase基因能够在乳腺上皮细胞中转录翻译并分泌到胞外。这些结果表明,所构建的人GlcCerase基因乳腺表达载体具有生物学功能,为下一步利用该载体进行转基因供体细胞的建立奠定了基础。第三部分奶山羊胎儿成纤维细胞的分离培养及脂质体法转染研究,采用组织块培养法结合胰蛋白酶消化法分离纯化得到奶山羊胎儿成纤维细胞,绘制了生长曲线,鉴定了胎儿细胞性别及核型特征,结果表明：该培养体系可以支持奶山羊胎儿成纤维细胞的体外生长,其细胞形态为梭形,高度汇合后呈火焰状,增殖特性以及核型特征均为正常,性别鉴定显示该奶山羊胎儿细胞为雌性,符合体细胞转基因克隆的基本要求。进一步利用脂质体法将pEGFP-Cl质粒转染入该细胞,研究了脂质体量、质粒量和转染时间对转染效率的影响,获得了脂质体转染该细胞的最佳条件：24孔细胞培养板中采用4.0μL脂质体转染试剂,1.2μg质粒DNA,细胞在复合物中孵育6 h。这为下一步目的基因转染奶山羊胎儿成纤维细胞的研究提供了参考依据。第四部分建立转人GlcCerase基因奶山羊胎儿成纤维细胞的试验,利用上述优化的转染体系,将线性化的乳腺特异性表达载体pBC 1-GlcCerase-Neo转染胎儿成纤维细胞,经G418筛选8-10天后,获得抗性细胞克隆,进一步通过96孔细胞培养板分离得到来源于单个转基因成纤维细胞的细胞克隆,经PCR扩增检测,得到稳定整人GlcCerase基因的转基因供体细胞8株,和对照组细胞比较,转基因过程中没有导致细胞的生长和核型异常。转基因细胞的核型(2n=58+XX)正常比例为66.8%,而第10-12代的非转染细胞核型正常率为70.9%,二者差异不显著(P>0.05)。这些结果表明可以利用这些阳性转基因细胞作供体细胞进行核移植研究。第五部分人GlcCerase转基因克隆胚胎的制备研究,采集屠宰山羊卵巢,获取卵母细胞并体外成熟培养,成熟率为63.3%。以100%汇合2天的转基因体细胞作核供体,以去核的MⅡ期卵母细胞作核受体进行核移植操作。完成核移植的卵母细胞采用122 kV/cm、20μs/次、间隔1s的直流电脉冲进行融合,融合率为83.3%,融合后的重构胚胎进一步用Ionomycin和6-DMAP进行激活处理,然后转移到SOFaa培养液中与单层卵丘细胞共培养,重构胚的卵裂率为89.1%,发育至桑葚胚/囊胚期的比例为36.4%。以正常体细胞来源的核移植胚胎作为对照,其融合率、卵裂率以及桑葚胚/囊胚率分别为77.8%、90.9%和38.9%,两种供体细胞来源的融合率和胚胎发育率差异不显著。手术法将发生卵裂且形态正常的转基因克隆胚(2-细胞期或以上)移植到同期发情的山羊输卵管中,16只受体山羊中有6只一直没有返情,在第40天经B超检测到2只妊娠的结果,但是最终妊娠没有发育到期,胎儿发生流产。第六部分探讨了外源基因转染对供体细胞生物学特性的影响。将构建的乳腺表达载体pBC1-GlcCerase-Neo分别转染体外培养的奶山羊乳腺上皮细胞、胎儿成纤维细胞以及成年皮肤成纤维细胞,获得整合有外源基因GlcCerase的转基因体细胞。以非转染的正常细胞为对照,利用流式细胞仪分析了转基因奶山羊体细胞的细胞周期分布和细胞凋亡的情况,研究结果显示：三种转基因细胞100%汇合2d后,G0/G1细胞的百分比都显著低于对照组细胞(P<0.05),其中转基因胎儿成纤维细胞的G0/G1期比例高于其它两种转基因细胞；转基因胎儿成纤维细胞凋亡率达22.56%,较对照组细胞有显著提高(P<0.05)；然后进一步利用荧光定量PCR法探讨了外源基因转染对胎儿成纤维细胞基因表达模式的影响,检测的基因分别为基因印记基因(IGF2, IGF2R)、凋亡相关基因(Bax)、应激相关基因(热休克蛋白,Hsp70.1)、细胞连接相关基因(Cx43)和DNA甲基化转移酶1基因(DNMT1)。其中IGF2, IGF2R和Cx43mRNA的转录水平显著高于对照组非转染的对照组细胞(P<0.05)。本研究首次从细胞周期分布、细胞凋亡以及基因表达变化模式等方面探讨外源基因转染对奶山羊体细胞的影响,探索转基因克隆效率低的内在机制,为更好地促进供体细胞的重编程,进一步提高转基因克隆的效率奠定了基础。更多还原

【Abstract】 Human lysosomal acidβ-glucosidase(G1cCerase) is responsible for the catabolism of glucosylceramide. Deficiency of GlcCerase activity results in the progressive accumulation of glucosylceramide, and it ultimately leads to clinical manifestations of anemia, hepatosplenomegaly, bonelesions, and in more severe cases, central nervous system impairment, which called Gaucher disease (GD) in clinical. GD is now treated mainly by administrating the missing enzyme (enzyme replacement therapy, ERT). Human GlcCerase is a difficult enzyme to be obtained, at present, CHO cells and transgenic plants systems are used for the production of recombinant G1cCerase, but which remian limited by their high cost in CHO cells and difficult purification and hypo-glycosylation in transgenic plants. Transgenic animal bioreactor offers particularly attractive possibilities to prepare recombinant G1cCerase, with the advantages of low cost production as well as high quality proteins. However, pronuclear microinjection have been used for more than two decades to produce transgenic animal, the low efficiency and high cost has been the main barrier for transgenic animal production. Nuclear transfer using transgenic donor cells has provided an alternative, efficient technique for the production of transgenic animals expressing a protein of interest. Therefore, we selected the human GlcCerase as the targeted object. Firstly, GlcCerase cDNA sequence was cloned and the biological activity of the protein was studied. Secondly, mammary-gland specific expression vector of GlcCerase cDNA was further constructed and the effectiveness of this vector was verified in in vitro mammary epithelial cells. Thridly, this mammary-gland expression vector was transfected into dairy goat fetal fibroblast cells, and stable transgenic cells integrated with G1cCerase gene were obtained by selection. Finally, the transgenic goat cloned embryos were produced by SCNT, in order to obtain transgenic goat mammary gland bioreactor expressing G1cCerase.The study was divided into six parts, the first and second parts focused on the cloning of G1cCerase gene, construction of GlcCerase expression vector and its expression in vitro, which provided basis for the expression of this vector in the mammary gland; the third parts and fourth parts were to optimize the transfection system of dairy goat fetal fibroblasts using liposome, and obtain donor cell lines stablely integrated with humanβ-glucosidase gene; the fifth part consists of using the transgenic cells as donor cells of nuclear transfer and evaluating the developmental ability of goat cloned embryos derived from these transgenic cells in vitro and in vivo. The sixth part was to identify the effect of GlcCerase gene transfection into dairy goat somatic cells in terms of cell cycle distribution, chromosome abnormality, incidence of apoptosis, and the relative abundance of gene expression. The main results were as follows:1. We amplified human GlcCerase gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T vector, the gene homology was 99% comparable to that of the reported human GlcCerase cDNA sequence in GeneBank, and one nucleotide difference was found, resulting in the change from aspartic acid to glycine. To test whether the cloned gene could encode the recombinant protein correctly, the GlcCerase gene was further subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-G1cCerase. After the recombinant plasmid were identified by restriction enzyme digestion, we transfected pEGFP-G1cCerase into COS7 cells by liposome, GlcCerase mRNA was expressed and the activity of GlcCerase was also detected in COS7 cells. These results indicated that the cloned human GlcCerase gene could encode proteins correctly and play a biological function, which could be used in the next step of mammary gland expression vector.2. The GlcCerase verified by the eukaryotic expression and the neomycin resistance gene (Neor) to permit selection of transformed cells were subcloned into pBCl vector containing goat beta-casein promoter respectively, then the constructed vector pBC1-GlcCerase-Neo was then proved by enzyme digestion and PCR amplification. In order to analyze the bioactivity of the vector, pBC1-G1cCerase-Neo was transfected into mouse mammary epithelial cell line HC-11. Positive single clone cells were selected with G418 and by PCR. The transgenic cells were cultured in induction medium containing RPMI-1640 medium with prolactin, insulin and hydrocortisone, which could induce recombinant human GlcCerase expression. RT-PCR and Western-blotting analysis showed that goatβ-casein gene promoter could regulate the transcription and translation of human GlcCerase gene in mammary-gland epithelial cells.These results indicated that the constructed expression vector of pBC1-G1cCerase-Neo had biological function, and could be used for further establishing transgenic donor cells with human GlcCerase gene.3. In order to prepare donor cells for dairy goat transgenic cloning, goat fetal fibroblasts cells (gFFCs) were isolated by attaching tissue explants from a day 30 goat fetus and purified by trypsin. The gFFCs were examined by cell morphology, growth curve and karyotype of chromosome, sex-determined region Y gene (SRY) of the gFFCs was also identified, which indicated that it’s suitable for the need of transgenic clone. Important factors involved in cationic liposome mediated gene transfer were also evaluated through in vitro transfection of gFFCs:the concentration of DNA and liposome, the effect of transfection time on the efficiency of gFFCs to express a reporter gene (GFP). The results showed that gFFCs cultured in 24-well culture plates with 4.0μL liposome and 1.2μg plasmid DNA for 6 hours resulted in the highest transfection efficiency, which was 4.21%. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer.4. The dairy goat fetal fibroblasts were transfected with linearized plasmid pBC1-G1cCerase-Neo using liposome by the optimized procedure and selected with G418. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates. The expanded clones were identified by PCR, the results indicated that the transgene was stably integrated into the open region of the chromatin of G418 resistant fibroblast cells. And the transgene did not result in the abnormalities of cell growth and chromosome ploidy, the percentage of transgenic cells with normal chromosomal number (60) were similar (66.8±3.2%) to those in control cells (70.9±0.8%, P>0.05). The above results indicated that these transgenic cell clones may be competent as donor cells for creating a transgenic goat by SCNT.5. Goat ovaries were obtained from a local abattoir and the cumulus-oocyte complexes (COCs) were matured in vitro,63.3% COCs were matured. Following nuclear transfer, stable fibroblast cell lines and non-transfected cells as control synchronized in Go by fully confluency for two days were used as donor cells respectively, MⅡoocytes without nuclear and first polar bodies were used as receptor cells. These reconstructed embryos were fused for 20μs under 1.2 kv/cm voltage, and activated using ionomycin and 6-DMAP, then co-cultured with cumulus cells in SOFaa. Similar rates (P>0.05) of fusion (83.3 vs 77.8%), and developmental capability for 2-4 cells (89.1 vs 90.9%),8-16 cells (50.9 vs 53.2%) and morula/blastocyst rates (36.4 vs 38.9%) were found between these two groups. Moreover, a total of 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients with two goats showing pregnancy at day 40. Unfortunately, the pregnancies failed to maintain to term, and the two fetuses aborted.6. Our study attempted to indentify the effect of gene transfection of human G1cCerase gene into dairy goat somatic cells on the characteristics and the relative abundance of gene expression, pBC1-G1cCerase-Neo plasmid were transfected into goat fetal-derived fibroblast cells (FFC), mammary epithelial cells (MEC), and adult ear skin-derived fibroblast cells (AEFC) respectively, and then transgenic somatic cell lines integrated with G1cCerase genes were obtained.The results showed that lower percentage (P<0.05) of cells at G0/G1 in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by the flow cytometric analysis. Further, we also examined the expression of genes involved in imprinting (IGF2, IGF2R), apoptosis (Bax), stress (heat shock protein, Hsp70.1), cellular connections (CX43) and DNA methylation (DNMT1) in transgenic T-FFC. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared to non-transfected FFC.This is the first study evaluating the influence of foreign gene transfection on dairy goat somatic cells in terms of cell cycle distribution, chromosome abnormality, incidence of apoptosis, and the relative abundance of gene expression, for exploring the inherent mechanism of low efficiency in transgenic cloning, and promoting the programming of donor cells and further improving the efficiency of transgenic cloning.更多还原