无血清条件培养基法诱导人胚胎干细胞成软骨分化的研究

发布时间：2018-04-05 22:31

本文选题：胚胎干细胞　切入点：软骨　出处：《吉林大学》2015年硕士论文

【摘要】：关节软骨作为关节中至关重要的部分，具有缓解关节面摩擦，减轻骨骼运动压力等重要作用。但由于其缺乏自我修复的能力，旦遭受外界创伤或衰退将造成关节软骨损伤、骨关节炎等软骨疾病，很难治愈。干细胞移植技术为软骨疾病的修复带来希望。人胚胎干细胞(humanembryonic stem cells，hESCs)由于其无限的增殖能力及多向分化潜能等优点，作为种子细胞明显优于成体干细胞或祖细胞；其个体差异小的优势使其能够快速建立分化平台，同时为iPSCs的相关研究奠定基础。通过体外诱导hESCs成软骨分化可以获得适宜的软骨组织及足够数量的软骨细胞,为软骨修复提出有别于传统治疗的新途径。在本课题中，我们采用两个阶段对hESCs进行软骨分化：中胚层分化阶段和从中胚层向软骨分化阶段。分化过程应用无血清的条件培养基，采取多种生长因子及小分子的组合。hESCs来源的软骨中胚层分化的条件培养基中主要包括CHIR (种高效、特异的GSK-3抑制剂，主要作用是激活WNT信号通路)，Noggin (BMP通路抑制剂)；后期从中胚层细胞向软骨分化时应用3D微球法培养，并结合了对软骨分化有促进作用的因子PDGF、 TGFβ、BMP等。3D微球培养后，细胞显示了显著的软骨相关性质，形成了透明软骨样球状团块。在分化过程中，从形态变化、基因水平、蛋白水平、组织学染色等方面进行了体外检测，并通过体内移植实验进步验证形成的软骨细胞，从而评价本课题方法对未来软骨修复的可应用性。实验结果显示，经过中胚层分化，细胞主要表达MEOX1转录因子并显示KDR-PDGFRα+表型,证明细胞经过轴旁中胚层向透明软骨方向分化；后期软骨分化检测结果表达SOX9等软骨祖细胞基因及COL2、COMP等软骨基质基因，，免疫组化显示COL2+COL1-,阿利新蓝、甲苯胺蓝等结果表明基质含丰富的蛋白多糖，这些鉴定说明在体外无血清的诱导条件н hESCs向透明软骨方向分化；通过细胞因子组合分化条件的探索发现，中胚层分化后若持续添加BMP4会导致阿尔新蓝染色变浅，显示基质中蛋白多糖含量减少п利于软骨分化；将分化的软骨细胞或软骨组织移植入免疫缺陷鼠体内，仍然能够维持软骨表型，并发现细胞在体内进步分化成熟。综т所述，课题初步证明了通过无血清条件培养基可以诱导hESCs成软骨分化，并取得理想的分化结果和较高的分化效率。这п仅为胎肝干细胞在体外进行软骨诱导提供新的实验依据，也为未来应用干细胞疗法治愈骨关节炎、关节软骨损伤等软骨疾病奠定基础。
[Abstract]:Articular cartilage, as an important part of joint, plays an important role in relieving joint surface friction and bone movement pressure.However, due to its lack of self-repair ability, suffering from external trauma or decline will cause cartilage injury, osteoarthritis and other cartilage diseases, it is difficult to cure.Stem cell transplantation is promising for the repair of chondrocytes.Human embryonic stem cells (HSCs) are superior to adult stem cells or progenitor cells as seed cells because of their infinite proliferative ability and multidirectional differentiation potential.At the same time, it lays a foundation for the research of iPSCs.Proper cartilage tissue and a sufficient number of chondrocytes can be obtained by inducing hESCs chondrogenic differentiation in vitro, which provides a new approach to cartilage repair, which is different from the traditional treatment.In this study, we used two stages of cartilage differentiation in hESCs: mesodermal differentiation and mesodermal to cartilage differentiation.In the process of differentiation, serum-free conditioned medium was used, and the conditional medium for differentiation of cartilage mesoderm derived from the combination of various growth factors and small molecules, HESCs, mainly included CHIR (highly efficient and specific GSK-3 inhibitor).The main role was to activate the WNT signaling pathway, the inhibitor of the Noggin WNT pathway, to culture the mesoderm cells into cartilage by 3D microspheres at the late stage, and to combine with the factors of promoting cartilage differentiation, such as PDGF, TGF 尾, BMP and so on, after cultured with 3D microspheres.The cells showed significant chondrogenic properties and formed hyaline chondroid bulbous masses.In the process of differentiation, the changes of morphology, gene level, protein level and histological staining were detected in vitro, and the developed chondrocytes were verified by in vivo transplantation experiments.Therefore, the application of this method to cartilage repair in the future is evaluated.The results showed that after mesodermal differentiation, the cells mainly expressed MEOX1 transcription factors and showed the phenotype of KDR-PDGFR 伪, which proved that the cells differentiated into hyaline cartilage through the paraxial mesoderm.Chondroid progenitor cells such as SOX9 and cartilage matrix gene COL2COMP were expressed in later stage chondrogenic differentiation assay. The results of immunohistochemistry showed that COL2 COL1, alisin blue and toluidine blue contained abundant proteoglycans in the matrix.These results indicate that hESCs differentiates into hyaline cartilage under serum-free induction in vitro, and through the study of cytokine combination and differentiation conditions, it is found that adding BMP4 to mesoderm after differentiation can result in light staining of Arxin blue.The results showed that the reduction of proteoglycan content in the matrix was beneficial to cartilage differentiation, and the transplantation of differentiated chondrocytes or chondrocytes into immunodeficient mice could still maintain the cartilage phenotype, and found that the cells improved differentiation and maturation in vivo.In this paper, it is preliminarily proved that hESCs can be induced into cartilage differentiation by serum-free medium, and the ideal differentiation result and high differentiation efficiency can be obtained.It only provides a new experimental basis for cartilage induction of fetal liver stem cells in vitro and lays a foundation for the treatment of osteoarthritis and articular cartilage injury by stem cell therapy in the future.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.3

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