【作者】 王挺；

【导师】 张铭；

【作者基本信息】 浙江大学 ， 细胞生物学， 2006， 硕士

【摘要】 骨髓间充质干细胞（msenchymal stem cells,MSCs）是一种具有自我更新和多分化潜能的成体干细胞,并且由于较低的免疫原性,它将在细胞治疗和组织工程上拥有越来越广阔前景。寻找合适的胎牛血清替代物,在体外优质高效地扩增人骨髓间充质干细胞,避免动物病毒污染是当前研究的热点。本实验以兔为模型,初步探索了利用异体血清替代胎牛血清培养MSC的方法和效果。 本实验成功地培养了兔子骨髓间充质干细胞。通过胫骨结节,股骨大转子和股骨下端等部位穿刺,取得了足够数量的骨髓,用密度梯度离心法和全血法分离,用含10%胎牛血清的DMEM培养基悬浮,在37℃,含5%CO₂的培养箱中培养。15天左右得到了集落汇合的原代骨髓间充质干细胞,一直被传到20代。本实验还比较了来自成年兔和新生兔的骨髓间充质干细胞的生长曲线,认为来自新生兔的骨髓间充质干细胞所需倍增时间显著低于成年兔细胞。本实验培养的骨髓间充质干细胞能经地塞米松诱导,茜红素S染色成阳性,并能在脑组织提取物诱导下分化为神经样细胞,证明细胞保持了多向分化能力。 本实验初步探索了利用异体血清替代胎牛血清培养MSC的方法。5个成年新西兰大白兔颈动脉放血,4℃冰箱过夜后收集混合的异体清。用硫酸铵沉淀法去除了清中的抗体IgG,又用透析法除去了硫酸铵,并且加热灭活了补体。我们用含10%兔异体血清的DMEM培养基成功地培养了原代骨髓间充质干细胞。细胞传至12代后才丧失分裂能力。这种细胞同样保持了成骨细胞和神经样细胞的分化潜能。本实验还比较了兔自体血清,异体血清和胎牛血清对细胞增殖的差异,认为在自体血清作用下,MSC倍增时间最短,而胎牛血清和异体血清之间不存在显著差异。没有经过抗体去除和补体灭活的异体血清不能支持MSC增殖。 本实验还探索了新生兔多种组织提取物,包括肝脏,脑,心脏,肾脏,脾,和胸腺对MSC增殖和分化作用。各种组织在冰浴匀浆后,4000r/min离心5min收集上清,用无血清DMEM培养基稀释成200ug/ml,过滤除菌后添加5%胎牛血清用于细胞培养。结果显示肝组织提取物能促进MSC的增殖,而脑组织提取物能诱导MSC分化为神经样细胞。。 此外,在综述部分,本文详细描述了骨髓间充质干细胞最近3年内在培养方更多还原

【Abstract】 Mesenchymal stem cells( MSCs) are a kind of adult stem cell with self-renew and multilineage differentiation potential. Primarily owing to their low immunogencity, they will be wildly used for cell therapy and tissue engineering in the future. Finding suitable replacement for fetal bovine serum to culture human MSCs and avoid the risk from animals contamination is a hot spot today. In this paper, we have established a rabbit model to use allogeneic serum for the culture of MSCs.On the first part of the experiment, we have successfully separated and cultured the MSCs from rabbit marrow. We have got enough marrow from suitable sites and ficoll density gradient separation and whole -marrow culture method are used in our research. The prime cultures usually last 15 days. The cells then can be subcultured untill the 20th passage. The self-renew abilities of MSCs from new born rabbit and adult rabbit are compared and the cells from new born rabbit show greater expansion speed. The MSCs can be induced into osteoblasts and neural-like cells under certain conditions.At the second part of the experiment, we mixed serum from different rabbits. The Ig G is separated by ammonium sulfate precipitation and the complements are heat inactived. MSCs are cultured by these serum from prime passage to the third passage and induced into osteoblasts successfully. The self-renew abilities are also compared between cells cultured under fetal bovine serum, autologous serum and rabbit serum. We found the cells cultured under autologous serum expanded more rapidly than others. The difference between fetal bovine serum and rabbit serum are not show. Nature rabbit serum with Ig G and active complements do not support cell expansion .At the third part of the experiment, we studied many kinds of rabbit tissue (liver, brain, heart, kidney, spleen and thymus ) lysates and their function on the expansion and differentiation of MSCs. Liver lysates support the expansion of MSCs and brain lysates can induce the cells differentiate into neural-like cells.At the review part, this paper describes the development of the culture forMSCs during recent years. In this paper, we list many factors, including the age of the donors, the different solutions for isolation, the density for cell culture, the mediums and the surface materials for culture, and their effect on the cell expansion . Here we emphasize the development of different substitution, such as growth factors and human autologous serum, for fetal bovine serum (FBS) to avoid the risk from potential by virus. MSCs infected plasmids with telomerase gene have shown stronger expansion ability and multilineage differentiation potential. The development of perfusion culture systems will make the preparation of MSCs more suitable for clinical application.更多还原