孤雌胚胎干细胞向软骨细胞定向诱导分化及构建组织工程软骨的研究

Seed cell is one of the key factors that constrains the research and application of cartilage tissue engineering. Chondrocyte has limited source in the body， and may lose chondrogenic phenotype during in vitro expansion, due to its dedifferentiation character. Marrow mesenchymal stem cells(MSCs) could differentiate into chondrocyte,osteoblast and adipocyte, and have long been considered to be an optimised source for cartilage tissue engineering. However, engineering cartilage with MSCs may encounter vascularisation and mineralization afer in vivo implantation, and result in the failure of engineered cartilage. Embryonic stem cells(ESCs) are harvested from the inner cell mass of the blastocyst and have complete potential to differentiate into all the primary germ layers such as ectoderm, mesoderm, and endoderm. ESCs could offer a virtually unlimited source of cells for use in tissue repair and regeneration. Stepwise approach to generate non-tumorigenic and lineage-restricted chondrogenic cells from ESCs under defined conditions has been proposed, and successful repair of critical-sized joint osteochondral defects has also been reported by implantation of tissue enginerred cartilage ,using chondrocytes derived from ESCs.However, harvesting of ESCs needs to destroy embryos, and destruction of embryos has ethical and political limitation.Furthermore, cells derived from ESCs will also trigger immune reaction after implantation.. Parthenogenetic embryonic stem cells(pESCs)could be established from parthenogenetic activation of oocytes, and avoiding destroy of embryo makes pESCs less concerns with political and ethical issues. pESCs have the pluripotency and proliferation capacity similar to ESCs. Cells derived from pESCs also will cause less immune reaction as the surface proteins on these cells has less veriation. The ethical advantage of derivation from unfertilized parthenogeneticaly activated oocytes, combined with immunomatching advantage makes pESCs a very promising source of seed cells for cartilage tissue engineering.However, whether pESCs could be directed to differentiate into chondrogenic lineage,and whether chondrocytes derived from pESCs are suitable as seed cell to engineering cartilage, have not been reported.. In the current study, we proposed that pESCs could be directed to differentiate into chondrocyte and used efficiently and practically for cartilage tissue engineering. We will induce pESCs to differentiate into chondrocytes in confined chondrogenic induction condition. Cartilage of small pellet, grafts of predetermined shape and injectable graft would be fabricated. Chondrogenic phenotype stability, cartilage formation, and immunoreaction characters of the grafts would be systematically studied and evaluated by both in vitro and in vivo model.

种子细胞是软骨组织工程研究和应用的关键。软骨细胞来源有限，且体外扩增过程中容易去分化而丧失表型；骨髓间充质干细胞（MSCs）是组织工程种子细胞的重要来源，但应用MSCs构建的软骨体内移植后最终会出现血管化和骨化而导致修复失败；胚胎干细胞具有很强的增殖能力和软骨分化潜能，虽然其来源的软骨细胞已成功应用于软骨缺损修复，但应用时仍会受到免疫排斥反应和伦理学的限制。本研究拟使用孤雌胚胎干细胞（pESCs，指通过化学物质激活卵母细胞后，分离孤雌囊胚的内细胞团体获得的胚胎干细胞）诱导分化成为软骨细胞并构建组织工程软骨。首先扩增pESCs并向软骨细胞定向诱导分化，对其表型特征和免疫原性进行系统观察；进而用pESCs来源的软骨细胞构建软骨团块并进行异体移植，观察软骨组织形成、表型维持、排斥反应等变化；最终用pESCs来源的软骨细胞进行大块特定形态软骨和可注射软骨构建，深入评价其应用前景。

胚胎干细胞（ESCs），可以无限地增殖并分化为软骨细胞，用于软骨组织的再生和缺损修复。伦理学上的争议始终是ESCs在学术研究及临床应用的障碍。研究表明，来源于孤雌囊胚的干细胞——孤雌胚胎干细胞（pESCs）具有ESCs特有的全能性，可以向内、中、外三个胚层方向分化，形成各种类型的体细胞。由于pESCs仅存在母性基因，故而只含有正常胚胎干细胞一半的主要组织相容性复合体（MHC），具有低的免疫原性。与此同时，哺乳动物来源的孤雌囊胚缺乏充分发育的胚外组织，不能形成真正意义上的胚胎，不存在伦理学争议，为pESCs应用于软骨组织工程提供了可能。.在本文的研究中，我们拟将小鼠pESCs作为软骨组织工程种子细胞的来源，首先验证pESCs的全能性，从细胞增殖能力、干性基因表达水平、体内形成畸胎瘤的能力、体外形成拟胚体（EB）及EB的分化潜能等多方面系统比较了pESCs与ESCs的区别。随后，利用尚且处于未分化状态的pESCs进行高密度微团培养（micromass），配合加入诱导因子BMP-2和TGF-β，在体外直接、有效地将pESCs诱导分化为较为均一的软骨细胞。紧接着，采用团块（pellet）培养法，促进细胞的进一步分化成熟，21天后，形成了接近于球形的、乳白色、表面光滑、有一定机械强度的软骨样组织。最后，将形成的组织取材固定，石蜡包埋切片进行后续的检测。在该实验中，我们采用PCR、实时荧光定量PCR、特殊染色及免疫细胞荧光染色等多种检测手段，分别从基因及蛋白水平对诱导成效进行评估，结果证实：pESCs增殖能力强，与ESCs相比不存在显著性差异，体内体外环境下均可以分化形成各种类型的体细胞，是一种全能干细胞。应用micromass法定向诱导pESCs成软骨细胞的实验结果显示：随着诱导时间的延长，软骨细胞的相关基因表达水平升高，胞外基质也逐渐累积，最终可以形成软骨样组织。.本实验中，我们证实pESCs是一种全能干细胞，由它诱导得到的软骨细胞在体外具有长期的增殖能力，因此我们认为：pESCs可以为软骨组织工程的发展提供一种安全的、有效的、没有伦理学争议的新策略。