【作者】 张拓；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 硕士

【摘要】 体细胞核移植技术作为一种生产转基因动物的方法有其不可代替的优越性,只要供体细胞筛选严格,可以保证100%的克隆后代表达外源基因。为了保证核移植效率,一般取低世代数(少于30 PDs)的动物体细胞作为核供体。但由于一些转基因方法,如基因打靶等,虽然可以将外源珍贵基因定点整合到阳性细胞基因组中,但效率较低,阳性细胞需要经历正负双重筛选阶段,而且为获得高效表达外源基因的阳性细胞还需经历复筛阶段,因此阳性细胞在体外培养需要经历较长的时间,导致最终获得的阳性细胞生长活力下降,不能满足核移植试验的要求。用于生产转基因动物的核供体细胞至少应保持旺盛的长势至45 PDs。而早期胎儿来源的细胞与其它动物体细胞一样,随着分裂次数的增加,染色体末端端粒结构进行性缩短,当端粒缩短到一定阈值时,便丧失了保护染色体序列完整性的功能,导致染色体稳定性下降,编码区序列丢失,进而发生融合或降解,最终导致细胞衰老死亡。因此,如何延长核供体细胞在体外培养的寿命,甚至使其永生化成为生产转基因动物的一个根本需求。研究发现,所有的配子细胞、干细胞以及近90%的癌细胞均表达端粒酶。这些细胞能够以端粒酶携带的RNA序列为模板,不断合成染色体末端端粒DNA序列,从而弥补细胞在分裂中端粒结构进行性缩短现象,延长细胞的寿命。至1998年,Bodnar等首先通过重建细胞端粒酶活性延长了正常人类细胞的寿命以后,不同动物来源的多种类型细胞均可在端粒酶的诱导下延长体外培养的寿命。但没有关于牛胎儿成纤维细胞(Bovine Fetal Fibroblast Cell,BFF)的报道。研究证明端粒酶逆转录酶(Telomerase Reverse Transcriptase,TERT)是端粒酶活性的限速成分,外源性人端粒酶逆转录酶(Human Telomerase Reverse Transcriptase, hTERT)基因导入多种正常体细胞后,能够激活端粒酶活性,使细胞的寿命延长,甚至永生化。本试验为获得一株生长性状稳定且具有优良遗传背景的荷斯坦奶牛成纤维细胞系,将含有hTERT基因的真核表达载体pCI-neo-hTERT通过脂质体介导法导入荷斯坦牛胎儿成纤维细胞。对转染后获得的阳性细胞进行周期检测,凋亡检测和倍体分析,并首次将表达hTERT基因的牛胎儿成纤维细胞作为体细胞核移植(Somatic Cell Nuclear Transfer, SCNT)供体细胞进行核移植试验。获得以下结果:(1)本实验利用手术法采集了妊娠46日龄荷斯坦奶牛胎儿,并分离、纯化了胎儿成纤维细胞。利用Lipofectimain 2000 Reagent将携带hTERT基因的真核表达载体pCI-neo-hTERT导入第5代胎儿成纤维细胞中,通过G418初筛和复筛,获得了表达hTERT基因的牛胎儿成纤维细胞,该细胞在体外培养至第50代时依然具有旺盛的生长活力;(2)对阳性细胞进行了周期检测、凋亡检测和倍性分析检测,结果显示:阳性第30代细胞与正常第30代细胞相比,处于S期细胞由19.59%上升至53.52%,正常第50代细胞开始出现凋亡现象,处于S期细胞仅占8.15%,而阳性第50代细胞仍保持较高活力(S=57.00%);正常第5代细胞与阳性第50代细胞凋亡率分别为:4.2%和4.5%,差异不显著,而阳性第50代细胞凋亡率为30.2%,与前两者差异极显著;阳性第50代胎儿成纤维细胞与正常第5代胎儿成纤维细胞具有同样的染色体核型。(3)试验中以阳性第50代细胞、正常第5代细胞和正常第50代细胞分别作为核供体进行了核移植试验,对克隆胚胎发育结果统计显示:以阳性第50代细胞和正常第5代细胞作为核供体获得的克隆胚胎囊胚率分别为21.33%(16/75)和24.10%(20/83),而以正常第50代细胞作为核供体获得的克隆胚胎囊胚率只有6.93%(7/101),统计分析显示,以正常第50代细胞作为核供体的克隆胚胎囊胚率显著低于前两者(P<0.01)。本试验结果表明:hTERT基因导入牛胎儿成纤维细胞后,可以延长细胞在体外培养的寿命并具有稳定的生物学特性,以其作为核移植供体的克隆胚具有正常的发育潜能,为生产转基因克隆动物提供了一株良好的核供体细胞。更多还原

【Abstract】 Technology of somatic cell nuclear transfer (SCNT) is an excellent method in producing transgenic animals and each transgenic animal produced by SCNT expressed external gene if the donor cell was screened strictly. In order to improve the efficiency of SCNT, the somatic cells with low population doublings (less than 30) are often chosen as the donor cells. Some kinds of advanced transgenic methods, such as gene targeting, could introduce the external interesting gene to a special site of genome exactly. However, the efficiency is low and often cost a long time to obtain a positive cell line for the positive cells expressing exogenous gene highly need to be screened by three main stages: the positive screening stage, the negative screening stage and the re-screening stage. The viability of positive cells will become lower with the process of selecting, so as to reduce the efficiency of SCNT.The donor cell used to produce transgenic animals should keep its excellent vigor to 45 population doublings at least. The bovine fetal fibroblast cells (BFF) are same as other kinds of somatic cells. The sequences of telomere at the end of each chromosome are shortened gradually during proliferation and if the lost sequences reach some threshold value, the telomere would lose its ability to keep the chromosome steadily. Then the code sequences (CDs) of some functional gene at the end of chromosome would also lose and the chromosome itself would be amalgamation or degradation easily. And finally, the cells are going to be consenescence and death. So prolonging the donor cells’lifetime cultured in vitro or enduring the donor cells to be immortalization are becoming a kind of essential requirement when producing trangenetic animals.All of the germ cells, the stem cells and nearly 90% of the carcinoma cell are positive of telomerase [6]. These cells can synthesis telomere DNA sequence at the end of chromosome spontaneously using its own RNA sequence as the template, so the lost telomere DNA sequence during cell proliferation would be remedy gradually, and the lifetime of these cells would be extened also.Since Bodnar (1998) extended the lifetime of normal human cells by introduction of telomerase, a great deal of cells derived from different animals having been extended their lifetime already. But there is on report about BFF in this field. Many studies showed the activity of telomerase is controlled by telomerase reverse transcriptase (TERT). Introduction of external hTERT gene could enhance the activity of telomerase, extend the lifespan and even endure the normal cells into immortalization.To work out a reliable system for transgenic animal studies, it is necessary to establish a BFF cell line with extended lifespan, excellent background in genetic and consistent functions and characteristics. In this study, the plasmid pCI-neo-hTERT was transfected into BFF by Lipofection 2000 Reagent. After screening, the cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM, and the SCNT embryo cloned from hTERT transfected cells were also studied originally. Experimental results obtained as follows:1. Bovine fetal fibroblast cells were isolated and purified from a 46 days Hostan fetal obtained by operation. The vector pCI-neo-hTERT was transfected into 5th generation of BFF by Lipofection 2000 Reagent. After screening, the positive cells were expanded and cultured to passage 50 in vitro still with excellent vigor.2. The cell cycles, cell apoptosis and the karyotype of the positive cells were detected by FCM. The experimental results obtained as follows:(a) Compared with 30th generation of negative cells, the percentage of S stage of generation 30 positive cells increased from 19.59% to 53.52%; 50th generation of the positive cells still have the ability of proliferation with the S stage of 57.00% but in negative cells of passage 50, the S stage is only account for 8.15% and cell apoptosis phenomena could be detected;(b) The apoptosis rate of 5th generation of negative cells and positive cells of passage 50 are 4.2% and 4.5%, respectively. The discrepancy is not significant (P>0.05). The apoptosis rate of negative cells of passage 50 is 30.2%. The discrepancy between negative cells and positive cells in passage 50 is significant (P<0.01).(c) Both the 5th generation of negative cells and 50th generation of positive cells have normal karyotype.3. In this study, three groups of BFF were used as donor cells to produce SCNT embryos. And the embryo development was observed and analyzed by statistic software SAS 6.12. The blastocyst rate in group positive 50 (P50) and negative 5 (N5) are 21.33% (16/75) and 24.10% (20/83), respectively. While the blastocyst rate in group N50 is just 6.93% (7/101). The discrepancy between group N50 and P50 is significant (P<0.01).The findings above demonstrated that the hTERT gene was transfected into BFF successfully and the lifespan of positive cells was extended. The positive cells cultured in vitro remained normal biological characteristics and the cloned embryos derived from them have excellent developmental potential.更多还原