间充质干细胞成骨分化和紫杉醇诱导鼻咽癌细胞凋亡的可视化研究

发布时间：2018-01-23 09:11

本文关键词： 原子力显微镜淫羊霍苷人脐带间充质干细胞成骨细胞紫杉醇鼻咽癌细胞细胞骨架　出处：《暨南大学》2010年硕士论文　论文类型：学位论文

【摘要】： 本论文主要分为两部分：(1)利用原子力显微镜(AFM)研究淫羊霍苷(ICA)诱导人脐带间充质干细胞(hUCMSCs)成骨分化的作用机制。(2)利用AFM研究紫杉醇诱导鼻咽癌细胞(CNE-2)凋亡。本文第一部分：通过形态学,细胞表面标志物以及多向分化能力三个方法鉴定hUCMSCs。使用改良钙钴法对加入ICA诱导后的细胞进行ALP染色,确定ICA是否具有诱导hUCMSCs分化为成骨细胞的作用。通过ALP阳性细胞计数,确定最佳促进成骨分化的ICA添加浓度,使用相同浓度的ICA诱导hUCMSCs向成骨细胞分化.结果表明：(1)光学显微镜图像显示,培养了7天的细胞为长梭形,是典型的成纤维细胞形态；培养了10天的细胞极性排列,集落呈漩涡状。(2)流式细胞仪(FCAS)检测分离到的hUCMSCs表面抗原CD29、CD44、CD 105阳性表达,CD34、CD45和人白细胞抗原HLA-DR阴性表达。(3)ICA能增加间充质干细胞的ALP的活性,促进矿化结节的形成(P0.05)。ICA最佳促进成骨分化浓度为1×10-6mol／L。(4)AFM的图像表明淫羊霍苷在盖玻片上呈分散状分布(粒径分布在70±25nm左右),加入细胞共培养5天时在细胞表面上聚集并呈微米域分布(粒径分布在96±21nm左右)。培养时间为10天时在细胞表面出现200-300 nm的微米孔,培养时间为15天时细胞表面微米孔的深度变小,细胞膜开始愈合,表现出细胞胞吞作用的现象。与分化前相比较,hUCMSCs的细胞形态由长梭形变为方形,细胞膜的粗糙度增大,细胞表面有明显的小突触,是因为成骨分化后细胞内形成钙结节。此结果为研究淫羊霍苷诱导间充质干细胞的成骨分化的作用机制提供了一种直观的方法。本文第二部分：应用AFM对紫杉醇诱导鼻咽癌细胞(CNE-2)凋亡的研究。(1)首先,采用MTT法检测紫杉醇对鼻咽癌细胞株(CNE-2)的生长抑制性,结果表明,紫杉醇作用后,细胞的存活率显著下降,且呈现显著的剂量依赖性。(2)采用不同浓度的TritonX-100处理CNE-2细胞,并用AFM对其进行形貌性质的研究,结果显示1ml／L的TritonX-100可以有效地制备出细胞骨架,为下一步研究紫杉醇作用后细胞骨架的变化提供了一个实验依据。(3)用AFM对紫杉醇诱导12小时,24小时和对照组CNE-2细胞的形貌,超微结构和粗糙度进行测量。与对照组细胞比较,发现诱导12小时后,细胞的体积开始皱缩,细胞绒毛减少；诱导24小时后,细胞发生明显的皱缩,细胞绒毛消失。并且随着诱导时间增加,细胞膜粗糙度增大。(4)用AFM对1ml／L浓度TritonX-100处理100μg／ml紫杉醇诱导12小时的CNE-2细胞进行成像；结果显示,紫杉醇可以破坏CNE-2细胞的细胞骨架。原子力显微镜是研究细胞骨架的一种可视化的工具。
[Abstract]:This thesis is divided into two parts: 1) the mechanism of osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) induced by ICA (atomic force microscope AFM) was studied. (. 2) AFM was used to study the apoptosis of nasopharyngeal carcinoma cell line CNE 2 induced by paclitaxel. In the first part of this paper, we used morphology, cell surface markers and multidirectional differentiation ability to identify the hUCMSCs.The modified calcium cobalt method was used to detect the ALP staining of the cells induced by ICA. To determine whether ICA can induce the differentiation of hUCMSCs into osteoblasts. By counting the positive cells of ALP, the best concentration of ICA to promote osteogenic differentiation is determined. The differentiation of hUCMSCs into osteoblasts was induced by the same concentration of ICA. The results showed that the cells cultured for 7 days were fusiform and typical fibroblasts. After 10 days of culture, the hUCMSCs surface antigen CD29 and CD44 were detected by flow cytometry (FCAS). The positive expression of CD105 and the negative expression of CD34-CD45 and human leukocyte antigen HLA-DR could increase the ALP activity of mesenchymal stem cells. Promoting the formation of mineralized nodules (P0.05A. ICA) the best concentration of osteogenic differentiation was 1 脳 10 ~ (-6) mol / L 路L 路L ~ (4) (AFM). The particle size distribution is about 70 卤25 nm. After 5 days of co-culture, the cells were clustered on the cell surface and distributed in micron domain (particle size distribution was about 96 卤21 nm). When the culture time was 10 days, the micropores of 200-300 nm were found on the surface of the cells. When the culture time was 15 days, the depth of the micropore on the cell surface became smaller, the cell membrane began to heal, showing the phenomenon of cell endocytosis, which was compared with that before differentiation. The cell morphology of hUCMSCs changed from long spindle to square, the roughness of cell membrane increased, and the surface of the cell had obvious small synapses. The results provide an intuitive method for the study of the mechanism of osteogenic differentiation of mesenchymal stem cells induced by aridonin. In the second part of this paper, AFM was used to study the apoptosis of nasopharyngeal carcinoma cell line CNE-2 induced by paclitaxel. The growth inhibition of paclitaxel on nasopharyngeal carcinoma cell line CNE-2 was detected by MTT assay. The results showed that the survival rate of CNE-2 cells decreased significantly after paclitaxel treatment. In a dose-dependent manner, CNE-2 cells were treated with different concentrations of TritonX-100, and their morphology and properties were studied by AFM. The results showed that 1 ml / L TritonX-100 could effectively produce cytoskeleton. It provides an experimental basis for the further study of cytoskeleton changes after paclitaxel treatment. (3) the morphology of CNE-2 cells induced by paclitaxel for 12 hours and 24 hours with AFM. The ultrastructure and roughness were measured. Compared with the control group, it was found that after 12 hours of induction, the volume of the cells began to shrink and the villi of the cells decreased. After 24 hours of induction, the cells shrank obviously and the villi disappeared, and increased with the induction time. AFM was used to image the CNE-2 cells treated with 100 渭 g / ml paclitaxel at the concentration of 1 ml / L TritonX-100 for 12 hours. The results show that paclitaxel can destroy the cytoskeleton of CNE-2 cells. Atomic force microscopy is a visual tool for studying cytoskeleton.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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