诱导多能干细胞向表皮干细胞定向分化的初步研究

发布时间：2018-05-05 09:00

本文选题：诱导多能干细胞 + 表皮干细胞　；参考：《深圳大学》2017年硕士论文

【摘要】：烧伤是临床常见外伤性疾病,是因为热力、化学试剂、电力等因素造成的组织器官的损害症状。严重烧伤会造成器官功能衰竭,引发多种并发症,还可能会危害患者生命。皮肤是否能够再生与修复的关键在于存在皮肤表层的表皮干细胞。然而大面积或者深度烧伤往往会将皮肤本身的表皮干细胞破坏掉,从而导致创面修复的困难。传统治疗方案包括自体、异体或异种皮肤移植,但手术结果并不总如人意。皮肤创面修复需要组织工程构建的皮肤替代物,表皮干细胞则是皮肤替代物常用的种子细胞。然而表皮干细胞的纯化率低,不能大量获取。因此如何在体外大量扩增表皮干细胞则是皮肤组织工程研究的关键点。2006年日本科学家发现将c-Myc、Klf4、Sox2和Oct-3/4四个与胚胎干细胞多样性相关的转录因子导入小鼠胚胎成纤维细胞(Mouse embryonic fibroblast,MEF),获得具有类似胚胎干细胞多样性的细胞,并称之为诱导多能干细胞(Induced pluripotent stem cells,iPSCs)。由于iPSCs避免了胚胎干细胞导致的道德伦理与法律问题,而且容易获得细胞,因此成为了组织工程的重要细胞来源。近十年来,iPSCs已被证实能够通过运用细胞因子诱导的方法定向分化为表皮干细胞。在现有iPSCs定向诱导分化为表皮干细胞的方法中,有使用羊膜诱导的、改变培养环境的、频频更换培养基的等等,这些培养方法的局限性在于诱导分化方法的不可控性以及操作的繁琐。本论文研究致力于使用添加成分明确的诱导表皮干细胞分化培养基(Epidermal stem cell differentiation medium,ESCDM),以提高表皮干细胞分化以及增殖的效率。本论文中,我将使用倒置显微镜(Inverted microscope,IM)、免疫细胞化学技术(Immunocytochemistry,ICC)方法对人iPSCs(Human iPSCs,hiPSCs)定向诱导分化为表皮干细胞的过程进行初步的探讨和研究。在本实验当中,iPSCs使用mTeSR?1完全培养基培养,避免与动物免疫蛋白接触。iPSCs传代之后分为实验组与对照组,实验组在诱导分化iPSCs采用添加维甲酸(Retinoic acid,RA)和骨形成蛋白4(Bone morphogenetic protein 4,BMP4)的方法,而对照组则采用羊膜提取液(Amnion extract,AETRA)进行诱导的方法。两组每天更换分化培养基,使用表皮干细胞无血清培养基(Defined keratinocyte serum free medium,DK-SFM)作为分化培养基。最终获得的细胞经过形态结构观察与ICC进行鉴定。同时设立人正常表皮干细胞(Normal human epidermal keratinocytes,NHEK)作为对照组,设立为观察分化得到的表皮干细胞与正常人表皮干细胞的差异。结果发现,诱导分化第6天之后,实验组的表皮干细胞的增殖数目显著多于对照组,免疫荧光技术检测诱导第6、8、10、12天两组的表皮干细胞的细胞角化蛋白CK19以及α6整合素的表达量,实验组的CK19以及α6整合素的阳性表达细胞均显著多于对照组的,可以得知实验组的诱导表皮干细胞分化的效率高于对照组。同时与正常表皮干细胞比较发现,实验组的形态与特性并无明显差别,增殖速度与正常表皮干细胞相同。而AETRA培养的表皮干细胞形态不一致、少量细胞出现分化、CK19以及α6整合素的表达量也偏低。综上所述,使用添加RA和BMP4的ESCDM缩短了hiPSCs定向诱导分化为表皮干细胞需要的时间,提高表皮干细胞分化的效率以及增殖的速度,同时维持了正常表皮干细胞的形态与特征。
[Abstract]:Burn is a common clinical traumatic disease, which is a symptom of tissue damage caused by heat, chemical reagents, electricity and other factors. Severe burns can cause organ failure, cause a variety of complications, and may also harm the patient's life. The key to the regeneration and repair of the skin is the epidermal stem cells in the skin surface. A large area or deep burn often destroys the epidermal stem cells of the skin itself, which leads to the difficulty of repairing the wound. Traditional treatments include autologous, allogenic or heterogeneous skin transplantation, but the results are not always desirable. Skin wound repair requires tissue engineered skin substitutes, and epidermal stem cells are skin. However, the purification rate of epidermal stem cells is low and can not be obtained in large quantities. Therefore, how to amplify epidermal stem cells in vitro is the key point of skin tissue engineering in vitro..2006 Japanese scientists found that four transcriptional factors related to c-Myc, Klf4, Sox2 and Oct-3/4 were introduced into small number of transcription factors related to embryonic stem cell diversity. Mouse embryonic fibroblasts (Mouse embryonic fibroblast, MEF), which have cells that have the diversity of embryonic stem cells, are called inducible pluripotent stem cells (Induced pluripotent stem cells, iPSCs). IPSCs avoids ethical and legal problems caused by embryonic stem cells and is easy to obtain cells, thus becoming an organization. The important cell source of engineering. In the past ten years, iPSCs has been proved to be able to differentiate into epidermal stem cells by using cytokine induced methods. In the existing methods of inducing differentiation into epidermal stem cells by iPSCs, there are amniotic membrane induction, change of culture environment, frequency frequency replacement medium and so on, and so on. The limitation lies in the uncontrollability of the induction of differentiation and the cumbersome operation. This paper is devoted to the use of Epidermal stem cell differentiation medium (ESCDM) to improve the differentiation and proliferation of epidermal stem cells. In this paper, I will use inverted microscope (I). Nverted microscope, IM), immunocytochemical Technology (Immunocytochemistry, ICC) method to induce the differentiation of human iPSCs (Human iPSCs, hiPSCs) into epidermal stem cells. In this experiment, iPSCs uses mTeSR? 1 culture to avoid contact with animal immunoglobulin. For the experimental group and the control group, the experimental group was induced to differentiate iPSCs by adding retinoic acid (Retinoic acid, RA) and bone morphogenetic protein 4 (Bone morphogenetic protein 4, BMP4), while the control group was induced by amniotic membrane extraction (Amnion extract, AETRA). The two groups changed the differentiation medium every day, using epidermal stem cells without blood. The clear culture medium (Defined keratinocyte serum free medium, DK-SFM) was used as a differentiation medium. The final cells were identified by morphological structure and ICC, and normal human epidermal stem cells (Normal human epidermal keratinocytes, NHEK) were established as the control group, which was established to observe the differentiation of epidermal stem cells and normal people. The results showed that after sixth days of differentiation, the number of epidermal stem cells in the experimental group was significantly more than that of the control group. The immunofluorescence technique was used to detect the expression of keratin CK19 and alpha 6 integrin in the epidermal stem cells of the two groups on day 6,8,10,12, the CK19 of the experimental group and the positive table of the integrin of alpha 6. The efficiency of the induced epidermal stem cells in the experimental group was higher than that of the control group. At the same time, compared with the normal epidermal stem cells, it was found that the morphology and characteristics of the experimental group were not significantly different from that of the normal epidermal stem cells. The morphology of the epidermal stem cells in the AETRA culture was not consistent, and a small amount of epidermal stem cells were found in the experimental group. The cells were differentiated, and the expression of CK19 and alpha 6 integrin was also low. To sum up, the use of ESCDM adding RA and BMP4 shortened the time required by hiPSCs to induce differentiation into epidermal stem cells, increased the efficiency of epidermal stem cell differentiation and the speed of proliferation, and maintained the morphology and characteristics of normal epidermal stem cells.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R644;R318.08

【参考文献】