孤雌胚胎干细胞向成纤维细胞和表皮细胞的定向诱导及构建双层组织工程皮肤的研究

Skin regeneration is an important area of research in the tissue engineering (TE) field, especially for massive skin loss cases, where current treatments are yet not capable of inducing permanent satisfying skin regeneration. To achieve an effective healing, skin TE products must attach well to the wound bed, be supported by new vasculature, integrate with the surrounding host tissues, be nonimmunogenic, and be capable of self regeneration with minimum scar tissue, with reduced patient pain and discomfort, and yet importantly, manufactured with a good cost-benefit ratio. Cultured skin began when methods for harvesting keratinocytes from patients and proliferating the cells in vitro became available. While engineered skin substitutes represent significant advances in wound care, their use is not routine because of their high cost, limited effectiveness, and their inability to reconstitute skin appendages.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 enginered 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 skin tissue engineering.However, whether pESCs could be directed to differentiate into epidermal lineage,and whether keratinocytes derived from pESCs are suitable as seed cell to engineered skin, have not been reported. In the current study, we proposed that pESCs could be directed to differentiate into keratinocyte and used efficiently and practically for skin tissue engineering. We will induce mouse pESCs to differentiate into keratinocytes in confined skin induction condition. Epidermal phenotype stability, and immunoreaction characters of the grafts would be systematically studied and evaluated by both in vitro and in vivo model.

种子细胞是皮肤组织工程研究和应用的关键。构建双层TES需要用到表皮细胞和成纤维细胞,但原代培养的表皮细胞和成纤维细胞来源有限，而且细胞质量不稳定。胚胎干细胞具有很强的增殖能力和多向分化潜能，其来源的表皮细胞已被成功应用于皮肤缺损修复，但应用会受到伦理学和免疫排斥反应的限制。本研究将孤雌胚胎干细胞诱导分化为表皮细胞和成纤维细胞，进行双层TES构建研究。首先扩增小鼠pESCs并向表皮细胞和成纤维细胞诱导分化，对其表型特征进行系统观察，重点观察细胞的角质形成能力和FGF、EGF、PDGF、VEGF、TGF、IGF等生长因子的表达水平；进而用pESCs来源的表皮细胞和成纤维细胞构建双层TES，观察双层TES的生物学特征；最后进行异体移植，观察皮肤缺损修复、表型维持、排斥反应等指标，深入评价这种新型双层TES的应用前景。

胚胎干细胞(Embryonic stem cells, ESCs)是一种有前景的TES种子细胞的来源，相比于包皮来源的成纤维细胞，ESCs具有来源不受限、体外扩增速度快的优点，但应用时可能受到伦理学的限制。孤雌胚胎干细胞(Parthenogenetic embryonic stem cells, pESCs)来源于人工激活的孤雌囊胚，相对于正常的ESCs，不破坏活的胚胎，由于只具有母系的遗传物质，能大大减弱免疫排斥反应，因此是一种理想的种子细胞来源。.在本研究中，我们首先比较了pESCs和ESCs两种胚胎干细胞的全能性和EBs中细胞的分化能力，对 EBs的进行贴壁培养期望获得MSCs。进一步通过分子生物学的方法检测了所获得的MSCs的性质，结果显示pESCs具有和ESCs相同的特征和三胚层形成能力，所获得的MSCs具有多系分化的能力。然后用所获得的MSCs进行诱导培养，获得了具有成纤维细胞表型特征的细胞。最后将获得的具有成纤维细胞表型特征的细胞接种于胶原水凝胶中构建出具有一定形态和机械强度的TES，并在小鼠背部进行缺损修复实验，通过大体标本观察和组织学观察评价修复的效果，结果表明诱导获得的成纤维细胞构建的TES具有和小鼠成纤维细胞构建的TES相似的皮肤缺损修复能力，并且pESCs来源的成纤维细胞构建的TES的修复效果和小鼠成纤维细胞构建的TES的修复效果没有显著差异。.这说明pESCs能够被定向诱导成为具有成纤维细胞典型特征的细胞，是一种理想的 TES的种子细胞。