人表皮干细胞的分离和培养的研究

发布时间：2018-01-03 06:10

本文关键词：人表皮干细胞的分离和培养的研究　出处：《江西医学院》2005年硕士论文　论文类型：学位论文

【摘要】：目的研究表皮干细胞的体外分离、培养及鉴定方法,观察表皮干细胞在有血清和无血清培养基中的生长特性,并对其在体外的培养条件进行探索,以期在体外获得人表皮干细胞的良好增殖。方法①采用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞,并以人成纤维细胞条件培养基为基础制备表皮干细胞培养基,用以人表皮干细胞(Ⅳ型胶原快速粘附法富集分离)的体外培养,以角朊细胞培养为对照组。观察人表皮干细胞的细胞形态和一般生长状态,比较人表皮干细胞和角朊细胞的克隆形成率和克隆维持时间,同时采用免疫细胞化学法检测β_1整合素和K_(19) 的表达,鉴定表皮干细胞。②将富集分离出的人表皮干细胞分两组培养,分别加入同源成纤维细胞条件培养基配制的人表皮干细胞有血清培养基和无血清培养基,观察和比较在这两种培养基中的细胞形态、克隆形成率、生长曲线、β_1整合素和K_(19)的强阳性表达率。结果①在Ⅳ型胶原快速包被的培养皿中快速贴壁细胞经继续培养,可见细胞克隆数增多,两周后可见大克隆形成。与角朊细胞相比,表皮干细胞有更强的克隆形成能力并可形成较大克隆,两者的形成率分别为17.04±1.01%和8.72±0.73%,两者比较有显著性差异(P0.01);人表皮干细胞β_1整合素和K_(19)免疫细胞化学染色呈强阳性。②细胞形态学分析和生长曲线均表明,培养5 天前,无血清培养的人表皮干细胞增殖细胞数较有血清培养的多,第9 天达到细胞基本融合状态并出现接触抑制;而在有血清培养基中,人表皮干细胞则继续增殖,第11 天时达到生长峰值,随后进入平台期。克隆性分析表明,两种培养基培养的表皮干细胞均呈克隆性生长,但比较克隆数目和大小、克隆形成率和克隆持续时间,有血清组明显优于无血清组(P0.01)。细胞周期分析显示,人表皮干细胞在有血清培养基中G_0～G_1期细胞比例明显
[Abstract]:Objective to study the isolation, culture and identification methods of epidermal stem cells, observe the growth characteristics of epidermal stem cells in serum-free and serum free medium, and explore their culture conditions in vitro, in order to obtain a good proliferation of human epidermal stem cells in vitro.
Methods using the Dispase II enzyme and trypsin two step isolated keratinocytes and fibroblasts, and human fibroblast conditioned medium for preparation of epidermal stem cell culture medium, with human epidermal stem cells (type IV collagen rapid adhesion by enrichment) in vitro culture of keratinocytes training for the control group. Observation of cell morphology and growth status of epidermal stem of human epidermal stem cells and cloning of keratinocytes and clone formation rate of maintenance time, at the same time using immunocytochemical method to detect beta _1 integrin and K_ (19) expression and identification of epidermal stem cells. The enrichment and separation of the the human epidermal stem cells cultured and divided into two groups, respectively adding homologous fibroblast conditioned medium of human epidermal stem cells serum medium and serum free medium, observe and compare the two culture medium on cell morphology, The positive rate of positive expression of clone formation rate, growth curve, beta _1 integrin and K_ (19).
The fast coated culture dishes in the rapidly adherent cells by cultured in collagen, visible cell clones increased after two weeks showed a large colony formation. Compared with keratinocytes, epidermal stem cells have a stronger ability to form clones and formed large clones, the formation rate is respectively 17.04 and 1.01%. 8.72 + 0.73% of the two, there is significant difference between them (P0.01); human epidermal stem cells beta _1 integrin and K_ (19) immunocytochemical staining showed strong positive analysis. The cell morphology and growth of Qu Xianjun culture showed that 5 days ago, serum-free culture of human epidermal stem cell proliferation cell number of serum culture more than ninth days to reach the cells fusion and contact inhibition; and in serum medium, human epidermal stem cells continued to proliferate, achieve the growth peak of eleventh days, then into the platform. Clonal analysis showed that two The cultured cells showed clonal growth of cultured epidermal stem, but the number and size of clones, clone formation rate and clone duration, serum group was significantly better than that of non serum group (P0.01). Cell cycle analysis showed that human epidermal stem cells in serum medium G_0 and the percentage of cells in G_1 phase significantly

【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

【参考文献】