【作者】 申晓东；

【导师】 宋关斌；

【作者基本信息】 重庆大学 ， 生物医学工程， 2005， 硕士

【摘要】 骨髓间充质干细胞(Marrow Mesenchymal Stem Cells,MSCs)是中胚层来源的具有多向分化潜能的干细胞,存在于不同种属的动物体内(如鸡、鼠、兔、犬、人),主要分布在全身的结缔组织及组织器官中,以骨髓组织中的含量最为丰富。在不同的诱导条件下,MSCs 具有向成骨细胞、成肌细胞、成软骨细胞、脂肪细胞和基质细胞等中胚层细胞分化的能力,同时还可分化为外胚层的神经细胞和内胚层的肝细胞。随着组织细胞工程学和基因工程学的发展,利用MSCs 的多向分化的潜能,将其运用于组织工程、细胞因子替代治疗、基因治疗、细胞移植及器官移植等方面的研究已成为热点。然而,MSCs 在体内含量很少,骨髓中每10 万单核细胞中大约有1 个MSCs,且随着年龄的增长,MSCs 数量逐渐减少。显然,如此微量的MSCs 根本难以满足细胞工程的需要。因此,如何摸索一个适宜的体外纯化、扩增的培养条件,对于进一步研究MSCs 的生物学特性、诱导分化和细胞移植就显得尤为重要。体外调节MSCs 增殖的方法有很多种,其中力学刺激是一种重要的途径。近年来,力学因素对细胞和组织生长发育的调节一直是生物力学十分重视的研究领域,国内外学者已经在这一领域作了大量的研究工作,积累了丰富的研究经验。拉伸是力学刺激中的一种有效方法,文献显示:最佳刺激条件的拉伸能促进细胞增殖。但目前利用力学拉伸加载作用调节MSCs 增殖的研究尚未见详细文献报道,其机理更不清楚。本实验拟研制拉伸加载装置,选取大鼠作为实验材料,进行MSCs的原代分离、培养、鉴定。然后在一定条件下对MSCs 的进行拉伸刺激,研究分析拉伸刺激后MSCs 的增殖情况,从中找到在设定范围内,该拉伸装置刺激MSCs增殖的最佳条件,并为以后MSCs 在力学拉伸刺激下的定向分化及进一步研究打下良好基础。本实验的研究方法和主要研究内容包括:首先,研制一套符合实验要求的拉伸加载装置,初步设定拉伸参数,进行相关预实验检测;同时,选取成年大鼠作为实验动物,采用Percoll 梯度离心法结合贴壁筛选法分离提取MSCs,培养传代,进行相关鉴定;然后使用拉伸加载装置,在一定的条件下,对MSCs 进行拉伸刺激,采用MTT 法分析增殖影响,分子生物学相关技术分析与增殖相关的c-fos 基因的表达变化,从中研究不同的拉伸刺激条件对MSCs 增殖的相应影响,并总结出在设定的参数范围内,本实验研制的拉伸加载装置刺激MSCs 增殖的最佳拉伸条件。本实验的研究结果如下:更多还原

【Abstract】 Marrow mesenchymal stem cells (MSCs), which root in the mesoblast, have the potentiality of differentiating into many kinds of cells. MSCs exist in different genera, such as chick, rat, rabbit, dog, human being, and so on. And they exit in connective tissue and apparatus, especially in the bone marrow. MSCs can differentiate into osteoblast, chondroblast, adipoblast, tendonblast, myoblast, cardiomyoblast, neuron, etc, in different inducement conditions. For this reason, MSCs have drawn more and more attention, and be used in tissue engineering, wound healing, cell transplant and gene therapy with the development of tissue engineering and gene engineering. But the quantity of MSCs is very few in animal and human being. There is only one MSC in about 100 thousands mononuclear in marrow and the quantity will decrease with the age increase. Obviously, this quantity cannot fit to the demand of cell engineering. So it is very necessary and important to find one way to purify and expand MSCs in man-made circumstance for the research of biologic characteristics, inducement, differentiation, and transplant of MSCs. Now there are many factors in vitro that could affect the proliferation of MSCs, and mechanical stimulation is an importance way. In recent years, that is always one importance field of biologic mechanics, which is the effect of mechanical factor on the growth and develop of tissue and cells. And scientists have carried out many research on it, and found a lots of rules. Stretch is one effective way in mechanical factor, and it was reported that stretch based on the best condition could promote the proliferation of cells. There is no detailed report about the effect of stretch on proliferation of MSCs up to now, so we do some research on it. The methods and contents include that: First of all, a kind of stretch equipment was designed, and the parameters of stretch were confirmed, and some preliminary experiments were done. Secondly, MSCs were separated by centrifugation in Percoll solution from adult rat, and cultivated and identifed MSCs. Thirdly, the effect of stretch on proliferation of MSCs was investigated, MSCs were stimulated by stretch equipment in different conditions, then the best stimulative condition of this stretch equipment and the change of expression of c-fos gene were analyzed by MTT and RT-PCR technique. Results: 1. Design of stretch equipment A kind of stretch equipment was designed based on the theory of stretch and rounded membrane. This equipment is controlled by a SCM, displays by LED, and its parameters can be adjusted easily and the range is linear. The range of the frequency is from 0.1Hz to 1Hz, and the intensity is from 2% to 15%, and the time is be controlled by manipulator. In general it can fit to the demand of this experiment. 2. Isolation and cultivation of MSCs Mononuclear cells were separated by centrifugation in Percoll solution (density 1.073g/mL). Then they were seeded in DMEM-LG supplemented with 10% fetal calf-serum and 1% antibiotic-antimyotic solution at a concentration of 1 ×106 cells/cm2。Cells were maintained at 37℃in a humidified atmosphere camber containing 5% CO2. The medium was changed after 72h, nonadhersive cells were removed with changing of medium, and twice weekly thereafter. When the cellss reached nearly 90%confluence. Then cell surface antigens were detected, and the result shown that CD34 was negative while CD44 and Fibronectin and CollagenI were positive. G0/G1 phase of cell cycle was 93.7%, and suggested that most of cells are not in period of proliferation. Analyse of AKP activity proved that these cells could differentiate into osteoblast. All of above results proved that the cells which we isolated and cultured in this experiment are MSCs.. 3. Effect of stretch on proliferation of MSCs MSCs were stimulated by stretch with different time and different intensity while the frequency maintained 1Hz. It found that proliferation of MSCs could be regulated through stretch, and the effect of the stimulative time is more effective than that of stimulative intensity. The best condition is that the time is 60min, the intensity is 8%, and the frequency is 1Hz, then expression of c-fos gene in MSCs increased obviously. Experimental results suggested that the equipment that I designed could fit to the demand for my research, and MSCs could be achieved by centrifugation with Percoll solution, and proliferation of MSCs would be promoted by stimulation of stretch.更多还原