【作者】 王敏力；

【导师】 李芳；

【作者基本信息】 大连医科大学 ， 免疫学， 2005， 硕士

【摘要】 目的: 探讨人体细胞核移植来源的兔-人胚胎干细胞(SCNT-rhESCs)体外保持未分化状态的扩增能力及其人胚胎干细胞特性,着重探讨 SCNT-rhESCs 在体外向成骨表型细胞分化的潜能和条件,尝试为骨组织工程探寻一种含有人自体基因、无免疫排斥反应、具有高分化潜能的全新种子细胞。 方法: 应用已建立的人体细胞核移植来源的兔-人胚胎干细胞(SCNT-rhESCs)系体外扩增培养 50 代后,进行系统的干细胞特性鉴定并进行体外成骨定向诱导分化研究。采用机械法收集 SCNT-rhESCs,首先在体外悬浮培养形成含有三个胚层(内、中、外胚层)定向祖细胞的拟胚体(Embryoid body,EB)。7 天后,将拟胚体转入细胞培养板内进行贴壁培养,分组进行成骨定向诱导研究:一组为成骨诱导组,采用含抗坏血酸(50mg/L)、地塞米松 (1×10-8 mol/L)、β-甘油磷酸(10mmol/L)、15%胎牛血清的α-MEM 矿化培养基;另一组为非诱导组,采用仅含 15%胎牛血清的α-MEM 培养基;采用具有成骨能力的胎儿骨髓间充质干细胞(MSC)作为实验的阳性对照。各组细胞均置于 37℃,5%CO2饱和湿度的培养箱培养。采用倒置显微镜适时观测诱导过程中的细胞形态学变化;通过茜素红染色、碱性磷酸酶(Gomori-钙钴法)染色、S-P 法免疫组化及免疫荧光染色检测骨钙素(Osteocalcin, OCN)以及Ⅰ型胶原的表达以鉴定成骨诱导后的细胞表型。 结果: SCNT-rhESCs 在体外扩增培养 50 代后,仍能保持未分化状态并具有人胚胎干细胞特性。表现为:人胚胎干细胞特异性表面标志 SSEA-3、SSEA-4、TRA-1-60等阳性,而鼠胚胎干细胞表面标志 SSEA-1 阴性,细胞核型正常,DNA 微卫星遗传标志分析显示其基因型与供体细胞完全一致,并且 SCNT-rhESCs 可以在体外自发分化形成拟胚体。同时,拟胚体贴壁培养后在成骨诱导剂的作用下 2-3 周时有成骨表型细胞出现,表现为:倒置镜下细胞呈多伪足或鳞片样,能形成茜素红染色阳性的骨结节;成熟成骨细胞标志——碱性磷酸酶染色阳性;S-P 法免疫组化及免疫荧光染色示骨组织中的主要胶原蛋白成分——Ⅰ型胶原以及骨组织中的主要的非胶原蛋白成分、成骨晚期标志物——骨钙素均呈阳性,提示 SCNT-rhESCs 在体外经成骨诱导剂的定向诱导后可以产生稳定的成骨表型细胞。而非诱导组SCNT-rhESCs 则形态各异,在不同时间段内部分自发分化成为神经元样细胞、脂肪样细胞、肌肉样细胞以及其他未定型细胞,未观察到有明显的成骨表型细胞出现。 结论: 1.人体细胞核移植来源的兔-人胚胎干细胞——SCNT-rhESCs 可在体外进行扩增且能保持干细胞的未分化特性和多向分化潜能。 2.同时,在成骨诱导剂的作用下 SCNT-rhESCs 可以分化成为稳定的成骨表型细胞。 因此,由 SCNT-rhESCs 来源的成骨细胞可以作为胚胎早期骨发生、发育的研究模型并可为骨组织工程提供全新的包含人自体基因、无免疫排斥反应的新种子细胞。更多还原

【Abstract】 Objective: Nuclear cloning, which has also been accurately called somatic cell nuclear transfer (SCNT), involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate an embryo with a genetic makeup identical to that of the donor. In this study, an embryonic stem cell line----- (SCNT-rhESCs) was established from clonally reconstructed blastocyst by transferring human somatic nuclei derived from lymphocyte into healthy enucleated rabbit oocytes. The aim was to study the biological characteristics and osteogenic capacity of SCNT-rhESCs after subculture in vitro so as to explore the feasibility and application potential of SCNT-rhESCs-derived osteoblasts as seed cells for bone tissue engineering and clinical application. Methods: SCNT-rhESCs was established, cultured and expanded on MEF with reported protocols for hES cells before stem cell characteristic identification was carried out. After SCNT-rhESCs formed embryoid body in suspension culture for seven days, cells were transferred to cell culture dishes to allow attachment and cultured in mineral medium contained α-MEM with 15% refined fetal bovine serum and supplements as dexamethasone (1×10-8 mol/L),βglycerophosphate (10mmol/L) and ascorbic acid (50mg/L). Phenotype of cells after induction were identified and characterized with inverted microscope, Alizarin red staining, alkaline phosphatase staining by Gomori and immunofluorescence staining of typeⅠcollagen and osteocalcin (OCN) which show the main collagenous and noncollagenous components in bone matrix. Result: After continuous proliferation for more than 50 passages, SCNT-rhESCs displayed typical human ES cell morphology and maintained all human ES cell features, including positive staining for both ALP and cell surface markers including SSEA-3, SSES-4, TRA-1-60 and negative staining for mESCs surface maker SSEA-1 and were capable of differentiating into embryoid bodies in vitro. The cells maintained normal karyotypes and were proved genetically identical to the source cells. Mineralized cells from osteogenetic differentiation of SCNT-rhESCs can form bone nodules which could be clearly identified by Alizarin Red staining. Moreover, the major noncollagenous component of bone matrix osteocalcin, as well as the predominant collagenous component collagen type Ⅰ , were localized to the mineralized cells by immunohistochemistry and immunofluorescence detection. Conclusion: It has been demonstrated that SCNT-rhESCs have strong capacity for self-renew and tremendous potential for multilineage differentiation even after long term expansion and in vitro prolonged culture, and they could be induced to differentiate into mineralized osteoblasts under the influence of ascorbic acid,β-glycerophosphate and dexamethasone. The availability of human autologous embryonic stem cells ----SCNT-rhESCs and in vitro osteogenetic differentiation potential bring more light into cell-based regenerative medicine and thus minimized rejection and relative side effects of immunosuppressive medications and may provide a suitable seed cell source for bone tissue engineering as well as a suitable model for studying the molecular processes of osteoblastic development.更多还原