基于原子力显微术对干细胞分化与黑色素瘤细胞凋亡的研究

发布时间：2018-10-15 07:24

【摘要】：细胞的分化是个体发育的一个重要阶段,胚层细胞的分化导致组织形成、器官发生和系统建成,细胞的异常分化会导致癌变。细胞的衰老和凋亡也与人体健康有着极为密切的关系。为了深入了解正常细胞的分化和肿瘤细胞的凋亡,探索细胞分化与凋亡的作用过程和机制,本研究以猪脂肪前体细胞、猪精原干细胞(SSCs)、小鼠黑色素瘤细胞B16为模型,采用原子力显微术(AFM)、激光共聚焦显微术(LSCM)、流式细胞术(FCS)、透射电子显微术(TEM)、CCK-8法及Western-blot等方法,对分化及凋亡过程中细胞的表面形貌、内部结构、力学性能和相关蛋白进行检测,获得了以下具有创新性的成果： 1.深入探讨脂肪前体细胞向成熟脂肪细胞分化前后细胞在纳米水平的变化,应用AFM扫描发现细胞核周围出现孔洞,膜表面变得光滑,粗糙度减小颗粒大小降低,膜表面粘附力由(146.33+11.23)PN降低到(119.95+8.67)PN。硬度与杨氏模量方面也降低了约20%,分化后的成熟脂肪细胞膜流动性更强,更易变形。本研究首次获得脂肪前体细胞分化前后细胞的超微结构和力学性能,为理解体内能量平衡和肥胖疾病的发生及诊断提供新的实验依据。 2.首次运用AFM研究SSCs的多能性,分析SSCs向脂肪细胞分化后细胞形貌及内部结构、分子的变化。成脂诱导后,SSCs由立体球形变为扁平不规则的圆盘状,贴壁性能提高,内部出现大量脂滴,膜表面粗糙度及颗粒大小增加。粘附力由(47.31士19.75)PN增加至(64.64±1.44)PN,硬度、杨氏模量均升高。TEM扫描得到细胞内部线粒体、内质网及空泡大量增多。完全分化后,成熟脂肪细胞内部特异性分子PPARy与C/EBPa出现。总结来说,我们从定性及定量两个角度表征了SSCs成脂分化所产生的一系列变化。 3.以B16细胞为研究对象分析替莫唑胺(TMZ)对其致凋亡作用,发现TMZ对能有效抑制B16细胞的生长,50μMTMZ作用细胞时,凋亡率达到最大为34.6%。随着药物浓度的增加,细胞膜及细胞核都产生不可逆损伤,细胞膜电位、内部Ca2+浓度和Caspase-3蛋白含量也发生相应的变化。此工作首次推断TMZ对B16细胞膜和核的毒性,可能是药物造成细胞死亡的一个机制。综上所述,本课题采用原子力显微术为基础对细胞形貌、超微结构及力学性能变化进行探测,结合生物技术手段对分化及凋亡过程表征,可以直观的看到在这两个重要阶段中细胞的变化,为生命活动的深入研究提供方法和依据。
[Abstract]:Differentiation of cells is an important stage of ontogeny. The differentiation of embryonic cells leads to tissue formation, organogenesis and system development, and abnormal differentiation of cells leads to carcinogenesis. Cell aging and apoptosis are also closely related to human health. In order to understand the differentiation of normal cells and apoptosis of tumor cells, and to explore the process and mechanism of differentiation and apoptosis, the model of melanoma cell B16 of porcine adipose precursor cells and porcine spermatogonial stem cells (SSCs) of (SSCs), mice was used in this study. Atomic force microscopy (AFM), (AFM), laser confocal microscopy (AFM), (LSCM), flow cytometry (LSCM), transmission electron microscopy (FCS), transmission electron microscopy (TEM), CCK-8) and Western-blot were used to detect the surface morphology, internal structure, mechanical properties and related proteins of cells during differentiation and apoptosis. The following innovative results have been obtained: 1. The changes of adipose progenitor cells before and after differentiation into mature adipocytes were studied. AFM scanning showed that there were holes around the nucleus, the membrane surface became smooth, and the roughness decreased. Surface adhesion force of membrane decreased from (146.33 11.23) PN to (119.95 8.67) PN. The hardness and Young's modulus also decreased by about 20%, and the mature adipose cell membrane was more fluidity and deformation after differentiation. In this study, the ultrastructure and mechanical properties of adipose precursor cells before and after differentiation were obtained for the first time, which provided a new experimental basis for understanding the energy balance in vivo and the occurrence and diagnosis of obesity diseases. 2. The pluripotency of SSCs was studied by AFM for the first time, and the morphology, internal structure and molecular changes of SSCs after differentiation into adipocytes were analyzed. After fat-forming, SSCs changed from a three-dimensional sphere to a flat and irregular disk, and the adhesion property was improved, a large number of lipid droplets appeared inside the membrane, and the surface roughness and particle size of the membrane were increased. The adhesion force increased from (47.31 卤19.75) PN to (64.64 卤1.44) PN, hardness, and Young's modulus increased. Mitochondria, endoplasmic reticulum and vacuoles were obtained by TEM scanning. After full differentiation, specific molecules PPARy and C/EBPa appeared in mature adipocytes. In conclusion, we characterize a series of changes in adipogenic differentiation of SSCs from both qualitative and quantitative perspectives. The apoptotic effect of temozolidomide (TMZ) on B16 cells was analyzed. It was found that TMZ could effectively inhibit the growth of B16 cells. When 50 渭 MTMZ treated the cells, the apoptotic rate reached the maximum of 34.6%. With the increase of drug concentration, the cell membrane and nucleus were irreversibly damaged. The cell membrane potential, the concentration of Ca2 and the content of Caspase-3 protein also changed correspondingly. This work for the first time inferred that the toxicity of TMZ to the cell membrane and nucleus of B16 may be a mechanism of cell death caused by drugs. In conclusion, the changes of cell morphology, ultrastructure and mechanical properties were detected by atomic force microscopy (AFM), and the differentiation and apoptosis process were characterized by biotechnology. The changes of cells in these two important stages can be seen intuitively, which provides the method and basis for the further study of life activities.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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