【作者】 乐融融；

【导师】 高绍荣；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 端粒对于细胞保持染色体的稳定性和分裂能力有着重要的作用。在人类退行性疾病的发生中,端粒酶的活性降低或者缺失所导致的细胞端粒的逐渐缩短产生了非常重要的影响。体细胞重编程作为一种可能的再生医疗的手段,未来将对退行性疾病的临床治疗产生重要的影响。体细胞核移植(Somatic cell nuclear transfer, SCNT)和诱导型重编程(Induced pluripotent stem cell, iPS)是体细胞重编程最主要的两种技术。然而这两种技术重编程具有端粒功能障碍及相关病症的供体细胞的能力还有待探索。端粒延长对体细胞重获多能性有重要的意义。iPS细胞诱导过程中端粒酶是端粒延长的主要机制。iPS细胞诱导过程中端粒延长十分缓慢,iPS细胞在建系后还需要数代的细胞传代才能使端粒长度达到与胚胎干细胞相似的水平。最近的研究表明在iPS细胞诱导过程中过表达早期胚胎重编程因子Zscan4可显著提高iPS细胞端粒的延长效率和iPS细胞的诱导效率,同时显著提高所获得的iPS细胞的分化潜能。SCNT技术利用卵母细胞的重编程因子进行体细胞重编程,而iPS技术则利用的是有限的几个重编程因子。因此SCNT技术重编程具有端粒功能障碍及相关病症的供体细胞的能力有可能比iPS技术更强。本研究利用端粒酶敲除(Terc-/-)小鼠作为模型来验证这个推测。第三代Terc-/-小鼠表现出明显的与端粒缩短相关的缺陷,主要表型有体型减小、寿命缩短,以及很多重要的器官如小肠、脾脏发生提早萎缩。本研究分别建立了来自第二代(Generation2, G2)和第三代(Generation3, G3) Terc-/小鼠的核移植胚胎干细胞(Nuclear transfer embryonic stem cells, ntESCs)和诱导型多能干细胞(Induced pluripotent stem cells, iPSCs)。与Terc-/-iPSCs相比,Terc-/-ntESCs表现出更强的分化能力和自我更新能力。实验结果表明在端粒酶缺失的情况下核移植克隆胚胎的发育过程中端粒有显著的延长,而在iPSCs诱导过程中端粒的长度没有显著的变化。G3Terc-/-ntESCs的端粒功能较来源的体细胞有明显的改善,而G3Terc-/-iPSCs的端粒功能较来源的体细胞发生了进一步的损伤,表现为极短端粒和染色体末端连接的比例增加。此外,G3Terc-/-iPSCs的线粒体功能也发生了进一步的损伤。G3Terc-/iPSCs的线粒体呼吸能力下降,细胞内积累了大量的活性氧并导致线粒体基因组的突变频率增加。而在G3Terc-/-ntESCs中,这些线粒体的功能缺陷是有明显改善的。有趣的是,G3Terc-/-iPSCs的线粒体功能异常不是由PGC-1α的表达抑制引起的。但是在G3Terc-/-iPSCs的分化过程中,PGC-1α的激活被抑制影响了线粒体的成熟。而在G3Terc-/-ntESCs的分化过程中,PGC-1α的表达升高足以使细胞内的线粒体分化为成熟的线粒体。以上的实验结果表明在端粒酶缺失的情况下,核移植技术能激活不依赖于端粒酶的机制进行端粒延长,并且能显著改善线粒体功能缺陷。因此核移植技术重编程有端粒和线粒体功能缺陷的供体细胞的能力比诱导型重编程技术更强。将来的研究可利用核移植技术发现新的重编程因子,从而优化现在的诱导型重编程技术,并且提高来自端粒和线粒体功能异常的病人的iPSCs的质量。更多还原

【Abstract】 Telomeres play key roles in maintaining chromosome stability and cell replicative capacity. Progressive telomere shortening due to absent or insufficient telomerase activity plays important roles in driving degenerative pathologies in humans. Somatic cell reprogramming holds great promise in future clinical applications, especially in the treatment of degeneration disorders. Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) represent two major approaches for cell reprogramming. However, little is known regarding the ability of these two strategies to rejuvenate cells from donors with telomere dysfunction-related syndromes.Telomere re-elongation is of great importance for the acquisition of pluripotency during reprogramming. Telomere lengthening during iPSCs induction mainly relies on telomerase, the action of which is a very time consuming process, and iPSCs telomeres need postreprogramming to reach the length resembling that of ESCs. Recently, oocyte-derived factor Zscan4has been shown to dramatically elongates telomeres during iPSCs induction and thus improves the reprogramming efficiency and the quality of iPSCs. Therefore, we speculate that SCNT utilizing factors in occytes to reprogramming somatic cells may rejuvenate cells with dysfuntioncal telomeres in a manner superior to that of iPSCs technology with only a few reprogramming factors. Here, we utilized late generation telomerase-deficient(Terc-/-) mice as a model to probe this question. In the third generation Terc-/-mice, disease states associated with short telomeres become evident, with a reduced body size, a decreased life span and atrophy of multiple tissues such as small intestine, spleen and testicles. SCNT-derived embryonic stem cells (ntESCs) and iPSCs were successfully derived from second generation (G2) and third generation (G3) Terc-/-mice, and ntESCs showed better differentiation potential and self-renewal ability. Telomeres lengthened extensively in cloned embryos while remained or slightly increased in the process of iPSCs induction. Furthermore, G3Terc-/-ntESCs exhibited improvement of telomere capping function as evidenced by decreased signal free ends and chromosome end-to-end fusion events. In contrast, there was a further decline of telomere capping function in G3Terc-/-iPSCs. In addition to telomere dysfunction, mitochondria function was severely impaired in G3Terc-/-iPSCs as evidenced by oxygen consumption rate (OCR) decline, reactive oxygen species (ROS) accumulation and dramatically increased mitochondria genome mutations while these deficiencies were greatly mitigated in G3Terc-/-ntESCs. Interestingly, PGC-la expression appeared to be irrelevant to the mitochondrial dysfunction in G3Terc-/-iPSCs. However, impaired mitochondrial maturation in differentiating G3Tern-/-iPSCs was associated with a failure of PGC-1α reactivation, which was mitigated in G3Terc-/-ntESCs.In summary, this study demonstrates that SCNT is superior to transcription factors mediated reprogramming in rejuvenating somatic cells with telomere and mitochondria defects. The breakthrough recently achieved in human SCNT studies further suggests that the identification of novel reprogramming factors might greatly improve the current iPSCs technology and enhance the quality of human iPSCs derived particularly from patients with telomere and mitochondria defects.更多还原