人自体基因胚胎干细胞体外定向诱导成骨细胞分化的研究

发布时间：2018-03-14 09:03

本文选题：人胚胎干细胞　切入点：成骨细胞　出处：《大连医科大学》2005年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective:
To explore the source of human nuclear transplantation in rabbit human embryonic stem cells (SCNT-rhESCs) and its ability to maintain amplification of embryonic stem cell characteristics in an undifferentiated state in vitro, to study SCNT-rhESCs cells to differentiate into osteoblasts phenotype potential and conditions in vitro, find a gene containing human autologous bone tissue engineering, without immune rejection. With high differentiation potential of new seed cells.
Method:
The application of the established human somatic cell nuclear transfer from rabbit human embryonic stem cell line (SCNT-rhESCs) cultured in vitro after 50 generations, the system of stem cell characterization and in vitro differentiation of bone orientation. Collect SCNT-rhESCs by mechanical method, first suspended in vitro formation containing three germ layers (in., ectoderm) embryoid body oriented progenitor cells (Embryoid body, EB).7 days, the embryoid bodies into cell culture plates in adherent culture, grouping osteogenic induction study: a group of osteogenic induction group, with ascorbic acid (50mg/L), dexamethasone (1 x 10-8 mol/L), beta glycerophosphate (10mmol/L), alpha -MEM mineralization in 15% fetal bovine serum medium; another group of non induced group, using only a -MEM containing 15% fetal bovine serum medium; with osteogenic ability of fetal bone marrow mesenchymal stem cells (MSC) as the experiment The positive control groups. Cells were placed in the temperature of 37 DEG 5%CO2 saturated humidity incubator, timely observation training. The morphological changes of the cells during induction by inverted microscope; by alizarin red staining, alkaline phosphatase (Gomori- calcium cobalt method) staining, S-P immunohistochemistry and immunofluorescence staining of osteocalcin (Osteocalcin, OCN) and the expression of collagen I in the identification of a cell phenotype induced by bone.
Result:
After 50 generations of culture in vitro, SCNT-rhESCs can still remain undifferentiated and have human embryo.
The characteristics of stem cells. For human embryonic stem cell specific surface markers SSEA-3, SSEA-4, TRA-1-60 positive, and embryonic stem cell surface marker SSEA-1 negative cells, normal karyotype, DNA microsatellite genetic marker analysis showed that the genotype is consistent with the donor cells, and SCNT-rhESCs can differentiate spontaneously in vitro embryoid body. At the same time, the embryoid wall after cultured in considerate osteogenic agent under the effect of 2-3 weeks of osteogenic phenotype cells, showed: cells under inverted microscope showed many pseudopodia or scales, can form bone nodules alizarin red staining positive; mature osteoblasts -- alkaline phosphatase staining; S-P method of immunohistochemistry and immunofluorescence staining of collagen components are shown in the bone tissue of type I collagen and bone tissue in the main non collagen, bone markers osteocalcin were advanced Positive tip induced directional SCNT-rhESCs in vitro osteogenic inducer can produce bone phenotype cells into stable and non induced group SCNT-rhESCs is different in different period, part of spontaneous differentiation into neuron like cells, fat cells, muscle cells and other undifferentiated cells, was observed there appear obvious osteogenic phenotype of cells.
Conclusion:
1., rabbit human embryonic stem cells derived from human cell nuclear transfer, SCNT-rhESCs can be amplified in vitro and can maintain the undifferentiated and pluripotent differentiation potential of stem cells.
2. at the same time, the osteogenic agent under the action of SCNT-rhESCs can differentiate into bone cells into a stable phenotype. Therefore, SCNT-rhESCs derived osteoblasts can be used as early embryonic bone development and research, the model can provide a new gene containing autologous bone tissue engineering, without immune rejection of new seed cells.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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