【作者】 刘丹；

【导师】 何晓晓； 王柯敏；

【作者基本信息】 湖南大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 干细胞是一类具有高度增殖和多向分化潜能的细胞群体,近年来随着对干细胞特性认识的加深,其基础与应用研究都有了突破性进展。不过目前一些经典的技术已成为干细胞研究面临的重要瓶颈,新技术的引入对于推动干细胞相关研究具有非常重要的意义。纳米技术作为一门20世纪80年代末发展起来的新兴学科,为干细胞的研究提供了新的契机。作为纳米材料重要成员之一的二氧化硅纳米颗粒,由于易修饰、易合成等优点,在生物医学领域得到了广泛应用,有望为干细胞相关研究提供新的手段。本论文瞄准纳米材料作为载体这一前沿研究方向,在考察二氧化硅纳米颗粒与间充质干细胞干细胞生物相容性的基础上,进一步开展了二氧化硅纳米颗粒作为生物相容性载体用于间充质干细胞的增殖、分化以及体内示踪研究。主要内容包括以下三个方面：1、二氧化硅纳米颗粒与间充质干细胞的生物相容性研究以间充质干细胞为研究对象,初步考察了二氧化硅纳米颗粒与间充质干细胞的生物相容性。首先利用MTT实验考察一系列浓度(0.05 mg/mL～2 mg/mL)二氧化硅纳米颗粒对间充质干细胞存活率以及细胞形态的影响,结果表明二氧化硅纳米颗粒对间充质干细胞存活率的影响存在浓度依赖性,细胞存活率随着颗粒浓度的增大逐渐降低,但是当与细胞孵育的颗粒浓度在0.1 mg/mL范围内时,细胞存活率都维持在80%以上；而同一浓度的二氧化硅纳米颗粒对间充质干细胞存活率的影响则存在时间依赖性,即随着培育时间的延长,存活率降低,负面影响增大。选择对细胞存活率影响很小浓度下的二氧化硅纳米颗粒处理间充质干细胞,并对处理后的间充质干细胞进行成脂和成骨诱导,通过组织化学染色和定量结果表明,与对照组相比,纳米颗粒处理的间充质干细胞其成脂和成骨分化能力并没有发生明显影响。同时,电子显微镜结果显示,二氧化硅纳米颗粒被间充质干细胞摄取,而摄取大量颗粒后的间充质干细胞依然保持良好的形态,保持了完整的细胞器超微结构。2、二氧化硅纳米颗粒作为胰岛素载体用于间充质干细胞的成脂分化胰岛素是间充质干细胞成脂分化的诱导因子之一,本章在考察二氧化硅纳米颗粒与间充质干细胞生物相容性的基础上,研究了二氧化硅纳米颗粒作为胰岛素载体诱导间充质干细胞成脂分化的可行性。首先利用直接吸附和PMPI交联两种方法分别将胰岛素修饰到二氧化硅纳米颗粒表面,傅里叶红外转换光谱结果表明,胰岛素能够很好的修饰到颗粒上,且修饰到颗粒上的胰岛素稳定性良好；细胞实验表明,通过两种方法交联到纳米颗粒上的胰岛素其生物活性没有受到影响,能够很好的诱导间充质干细胞向脂肪细胞的分化,因此功能化二氧化硅纳米颗粒有望发展成为一种新型的干细胞诱导因子载体诱导间充质干细胞成脂分化。3、荧光二氧化硅纳米颗粒用于间充质干细胞的标记和体内示踪结合本实验室已有的二氧化硅荧光纳米标记技术平台,在二氧化硅纳米颗粒应用于肿瘤标记、活体示踪的基础上,初步探讨了荧光二氧化硅纳米颗粒对间充质干细胞的标记与体内示踪的可行性。利用流式细胞仪优化纳米颗粒标记干细胞的最佳浓度与时间,结果表明,1000μg/mL浓度的纳米颗粒与间充质干细胞孵育30min时,颗粒有效的进入细胞,细胞内的颗粒荧光强度达到饱和,表明荧光二氧化硅纳米颗粒可以有效的标记间充质干细胞；在此基础上,将标记的间充质干细胞以皮下注射和静脉注射两种方式移植裸鼠体内,利用活体成像仪成像,皮下注射处24 h内可以观察到荧光,标记的间充质干细胞经静脉移植后,将裸鼠解剖并对相关器官进行组织切片,结果在肝、肺、脾可以观察到荧光,初步推测间充质干细胞经过血液循环后可能迁移到了此部位,这对于研究干细胞的迁徙、归巢等生物学行为具有十分重要的意义。但如何改善纳米颗粒标记的间充质干细胞在体内的荧光强度,还有待于进一步研究。更多还原

【Abstract】 Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types. Recently, the wide spectrum of nanotechnologies holds great promise toward the study of stem cell biology and the development of new approaches to stem cell research. By combining the two important research fields of nanotechnology and stem cells research, the stem cells nanotechnology has been well developed and become a new intercross interdisciplinary field. Silica nanoparticles (SiNPs) are of interest for their biocompatibility and their mechanical properties. Because of their unique characteristics, SiNPs have been widely studied in a range of areas including chemistry, engineering and biomedicine, especially the application of functionalized SiNPs for cancer diagnostics and therapy. The nanoparticle’s good biocompatibility and easy modification make them well-suited for a variety of biomedical studies, which provides a new route for stem cells research. Aiming at the direction of nanomaterials based carriers, the biocompatible silica nanoparticles (SiNPs) based carriers for Rat mesenchymal stem cells (RMSCs) proliferation, differentiation and in vivo tracking have been reported. The three parts of the thesis are as follows.1. Bioeffects of silica nanoparticles on mesenchymal stem cells proliferation and differentiationThe cell viability assay of RMSCs in the presence of pure SiNPs was first performed using MTT assay and morphology test. The results indicated the addition of SiNPs to the cell culture medium with last concentration of 0.05 mg/mL and 0.1 mg/mL did not affect the metabolic activity and the proliferation of RMSCs. As the concentration of SiNPs increased, the cell survival rate decreased. If the last concentration of SiNPs was not higher than 0.1 mg/mL, the survival rate of the cells after exposure for 24 h still kept around 80%. the pure SiNPs with concentration of creating little cytotoxicity had no effect on the on cellular ultrastructures, adipogenic differentiation and osteogenic differentiation. Similarly, TEM analyses of RMSCs results indicated that the the structures of treated RMSCs were preserved by compared with the control group without treatment of SiNPs and stayed in a relatively active period with normal cell function.2. Silica nanoparticles as biocompatibile carriers of insulin for mesenchymal stem cells adipogenic differentiation In the present study, the SiNPs based carriers of insulin for RMSCs adipogenic differentiation have been reported. SiNPs were then selected as carriers to be conjugated with insulin, one of important growth factor supplements for RMSCs adipogenic differentiation. In vitro cell studies were carried out in order to evaluate adipogenic efficacy of adipogenic induction media containing SiNPs-insulin conjugates through the control experiments. The results showed that the biological activity of insulin conjugated to the SiNPs was not affected and the SiNPs-insulin conjugates could be used for RMSCs adipogenic differentiation, which would help to expand the new potential application of SiNPs in stem cell research.3. Fluorescent nanoparticles for stem cell labeling and stem cell in vivo trackingThe feasibility of cellular labeling and tracking in vivo of Rubpy silica nanoparticles (FSiNPs) in the RMSCs have been studyed based on the the front work of tumor labeling and tracking. FSiNPs in the cells were assessed by flow cytometry and the result showed that 1000μg/mL of FSiNPs could enter RMSCs efficiently after 30 min incubation.The FSiNPs labeled stem cells were firstly injected into the neck of null mice subcutaneously. There was fluorescence of FSiNPs for 24 h by using an optical in vivo imaging system. The labeled stem cells were further injected into the tail veins of null mice and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result showed that characteristic fluorescence of FSiNPs was observed in the liver, the lung and the spleen, which primarily presumed the migration of stem cells. The challenge should be how to improve the internalizing efficiency of FSiNPs in the stem cells.更多还原