诱导多能干细胞（iPS）的功能与其向黑素细胞分化能力的差异性研究

发布时间：2018-01-03 01:14

本文关键词：诱导多能干细胞（iPS）的功能与其向黑素细胞分化能力的差异性研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:诱导多能干细胞(iPS细胞)向黑素细胞的分化过程需耗费大量经费和极长时间,然而不同的i PS细胞功能差异很大,分化能力也各有差异,因此有必要对iPS细胞功能进行评估,从而选择更好的iPS细胞进行后期的分化研究。旨在探讨iPS细胞功能与其向黑素细胞分化能力的差异性。方法:(1)制备iPS细胞并选择多个iPS细胞株,检测并比较其AP染色强度、干细胞表面标志物、干性基因表达水平、体外拟胚体的形成以及畸胎瘤的结果;(2)在体外尝试将这些iPS细胞株向黑素细胞诱导分化,并对诱导的黑素细胞进行初步鉴定;(3)根据检测比较iPS细胞株的结果进行评估细胞的功能,并观察后期能否诱导出黑素细胞,从而研究分析iPS细胞功能与其向黑素细胞分化能力的差异。结果:(1)在碱性磷酸酶染色上,iPSC-1、i PSC-2和iPSC-3被碱性磷酸酶染色均呈阳性表达,表明iPSC都处于未分化状态。但其中iPSC-1碱性磷酸酶染色阳性强于iPSC-2和i PSC-3;(2)在干细胞表面标志物表达上,经细胞免疫荧光染色后iPSC-1、iPSC-2和iPSC-3均能表达干细胞表面标志物OCT4和NANOG,说明iPSC都具有干性。但在iPSC-1中,核定位的OCT4和NANOG荧光团比iPSC-2和iPSC-3更密集,表明其克隆内细胞更紧凑;(3)在干性基因表达上,iPSC-1、i PSC-2和iPSC-3均能表达Oct4、Sox2、Nanog、Rex1、GDF3、DNMT3B干性基因,表明iPSC都具有干性,且iPSC-1干性基因表达水平高于iPSC-2和i PSC-3,说明iPSC-1干性更好;(4)在体外拟胚体形成上,与i PSC-2和iPSC-3相比,iPSC-1体外第一天形成拟胚体的形态更圆、边界更清晰,第三天观察其体积更大,说明iPSC-1来源的拟胚体生长速度更快、体外分化能力更好;(5)在畸胎瘤形成上,将iPSC注入免疫缺陷型小鼠体内两个月后,iPSC-1可成功形成畸胎瘤,而iPSC-2和i PSC-3未能形成畸胎瘤,表明iPSC-1具有体内分化的多能性;(6)在体外定向分化为黑素细胞上,iPSC-1在第3周分化为黑素样细胞,通过细胞学形态和细胞沉淀颜色观察及黑素相关基因检测,证实iPSC-1成功分化为黑素细胞,而iPSC-2和i PSC-3分化过程中未形成黑素细胞。结论:不同的iPS细胞在功能上存在差异,其中碱性磷酸酶活性更高、干细胞表面标志物及干性基因表达水平更高、体外拟胚体形态更好以及体内可成功形成畸胎瘤的iPS细胞其功能更好,在体外能成功诱导分化为黑素细胞,即iPS细胞功能越好,则其向黑素细胞分化的能力越强。iPS细胞功能的差异会导致其定向分化为黑素细胞存在差异。
[Abstract]:Objective: to induce the differentiation of pluripotent stem cells into melanocytes, it takes a lot of money and a long time to differentiate into melanocytes. However, the function of different iPS cells is very different, and the ability of differentiation is also different. Therefore, it is necessary to evaluate the function of iPS cells. The purpose of this study was to explore the difference between the function of iPS cells and their ability to differentiate into melanocytes. IPS cells were prepared and several iPS cell lines were selected. The AP staining intensity, stem cell surface markers, dry gene expression, embryoid formation in vitro and the results of teratoma were detected and compared. (2) the iPS cells were induced to differentiate into melanocytes in vitro, and the melanocytes were preliminarily identified. The function of iPS cell line was evaluated according to the results of detection and comparison, and the melanocyte induction was observed at the later stage. The difference between the function of iPS cells and their differentiation into melanocytes was analyzed. Results: the iPS cells were stained with alkaline phosphatase (ALP). I PSC-2 and iPSC-3 were positive by alkaline phosphatase staining. The results showed that all iPSC were undifferentiated, but iPSC-1 alkaline phosphatase staining was stronger than that of iPSC-2 and I PSC-3. (2) in the expression of stem cell surface markers, both iPSC-1 and iPSC-3 could express OCT4 and NANOG after cell immunofluorescence staining. The results showed that both iPSC were dry, but in iPSC-1, the nuclear localized OCT4 and NANOG fluorescent groups were denser than those of iPSC-2 and iPSC-3, which indicated that the cloned cells were more compact. (3) both PSC-2 and iPSC-3 could express Oct4S0x2 NanogRex1 (GDF3) on dry gene expression. The dry genes of DNMT3B indicated that all iPSC had dryness, and the expression level of iPSC-1 dryness gene was higher than that of iPSC-2 and I PSC-3, which indicated that iPSC-1 had better dryness. (4) compared with I PSC-2 and iPSC-3, the formation of in vitro embryoid body was more round, the boundary was clearer and the volume was larger on the third day than I PSC-2 and iPSC-3. The results showed that the embryoid derived from iPSC-1 grew faster and had better differentiation ability in vitro. (5) in the formation of teratoma, two months after injecting iPSC into immunodeficient mice, iPSC SC-1 could successfully form teratoma, but iPSC-2 and I PSC-3 could not form teratoma. The results showed that iPSC-1 had pluripotency of differentiation in vivo. In vitro, iPSC-1 differentiated into melanocyte-like cells at the 3rd week. The morphology of cells and the color of cell sedimentation were observed and the melanin-related genes were detected. It was confirmed that iPSC-1 differentiated into melanocytes successfully, but no melanocytes were formed in the differentiation between iPSC-2 and I PSC-3. Conclusion: different iPS cells have different functions. The activity of alkaline phosphatase is higher, the expression level of stem cell surface marker and dry gene is higher, the morphology of embryoid body in vitro is better and the function of iPS cells which can successfully form teratoma in vivo is better. The better the function of iPS cells is, the stronger the ability to differentiate into melanocytes. The difference in function of IPs cells will lead to the difference of their directional differentiation into melanocytes. 3. The differentiation of melanocytes into melanocytes can be induced successfully in vitro, that is, the better the function of iPS cells is, the stronger the ability to differentiate into melanocytes.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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