大鼠毛囊干细胞的分离培养和鉴定方法研究

发布时间：2018-03-20 23:17

  本文选题：毛囊干细胞　切入点：分离培养　出处：《新疆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：众所周知,干细胞是指未成熟细胞,具有自我更新和分化能力,可分化为胚胎干细胞和成体干细胞、其他细胞、组织和器官。毛囊的周期性生长提示毛囊存存在着一群具有强增殖能力的细胞称之为毛囊干细胞。毛囊干细胞来源广泛且取材较容易；细胞在体外显示出很强的生长能力和集落形成能力,单个细胞可分裂增殖至1.7×1038个；不涉及伦理和道德问题的限制；能分化成表皮、皮脂腺和毛囊以及神经元、脂肪细胞和成骨细胞；因而成为组织工工工程理想的种子细胞。目前,毛囊干细胞的筛选纯化主要有两类方法：差速贴壁法和免疫磁珠法。其中,免疫磁珠法花费昂贵,操作复杂,筛选过程中可能会导致细胞活性受损,国内仅有少数有条件的科研单位能够完成,因而并非理想的筛选纯化方法。我们通过查阅国内外文献,利用我校现有的实验设备和技术平台,通过反复试验探索,联合使用显微分离技术、二步酶法以及差速贴壁法,亦可获得足量高纯度、活性好的毛囊干细胞。方法：经伦理学审查同意后,在实验过程中,选用清洁级SD大鼠的8-9天乳鼠作为研究动物。经过严格消毒大鼠乳鼠触须部组织后,手术取下含有毛囊的触须部组织。然后在超净台中,体式显微镜下,分离出大鼠触须部的毛囊组织,切取毛囊外根鞘部位；再分别使用中性蛋白酶和胰酶在自动摇床中消化；利用Ⅳ型胶原包被的培养瓶(即差速贴壁法)筛选出贴壁较快的细胞用10%胎牛血清的角朊细胞无血清培养基进行培养。所获细胞进行传代、消化以及冻存等技术处理。通过倒置显微镜定期观察照相、电子显微镜照相、姬姆萨染色、免疫荧光染色、绘制生长曲线以及流式细胞术检测等进行鉴定。结果：筛选后的细胞在倒置相差显微镜下观察形态均匀一致,折光性强,呈典型的“铺路石状”。透射电镜拍照显示细胞体积小,核浆比例大,细胞器发育不成熟,处于原始状态。绘制生长曲线显示,传代至P3-P6细胞具有较强的增值能力。流式细胞术所获得的数据通过统计学处理表明,所获细胞纯度可达90%以上。因此,我们的体会是联用显微分离技术、二步酶法以及差速贴壁法可获得纯度高、活性好的毛囊干细胞,联用CD34和β1整合素是较理想的鉴定方法。为进一步的诱导分化实验打下了良好的基础。
[Abstract]:Objective: as we all know, stem cells are immature cells that have the ability to renew and differentiate themselves into embryonic stem cells and adult stem cells, other cells, Tissue and organ. The periodic growth of hair follicle suggests that there exists a group of hair follicle stem cells with strong proliferative ability. Cells show strong growth and colony formation in vitro, with individual cells dividing and proliferating to 1.7 脳 1038; without ethical and moral limitations; differentiating into epidermis, sebaceous glands, hair follicles, and neurons. Adipocytes and osteoblasts; thus become ideal seed cells for tissue engineering. At present, there are two main methods for screening and purification of hair follicle stem cells: differential adherence and immunomagnetic bead, in which immunomagnetic beads are expensive. The operation is complicated, the screening process may lead to the damage of cell activity, only a small number of qualified scientific research units in China can complete, so it is not an ideal method of screening and purification. Using the existing experimental equipment and technology platform, through repeated experiments and exploration, combined use of microscopic separation technology, two-step enzymatic method and differential adhesion method, we can also obtain sufficient quantity and high purity. Methods: healthy hair follicle stem cells were obtained. Methods: the SD rats of clean grade were selected as the study animals for 8-9 days after the ethical review. After strict disinfection of the tentacles of the rats, the hair follicle stem cells of the SD rats were disinfected strictly, and the hair follicle stem cells of the SD rats were disinfected strictly. The tentacles containing hair follicles were removed by surgery. The hair follicle tissue of rat tentacles was isolated under a pose microscope and then digested in a shaker with neutral protease and trypsin, respectively, and the outer root sheath of the hair follicle was removed. By using the culture bottle coated with type 鈪，

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