脂肪干细胞增殖特性及向心肌细胞分化的研究

发布时间：2018-05-27 08:06

本文选题：脂肪干细胞 + 支架　；参考：《大连理工大学》2009年博士论文

【摘要】： 目前心肌梗死仍是发病率和死亡率较高的疾病之一。由于心肌细胞再生能力有限,坏死的心肌组织即使得到再灌注治疗也只能由无收缩功能的瘢痕组织替代,细胞移植治疗只能修复小面积心肌损伤,而组织工程技术为大面积心肌缺损提供了一个较好的治疗方法。组织工程包括种子细胞、支架材料和生物活性分子三大要素。干细胞由于具有自我更新和多向分化潜能将成为重要的组织工程种子细胞,但由于胚胎干细胞、骨髓干细胞以及诱导的多潜能干细胞存在着伦理道德或免疫排斥反应,甚至有致瘤的危险性,而脂肪干细胞由于来源简便充足,容易大量提取获得,移植后无免疫排斥反应,将成为组织工程比较有前景的种子细胞。因此我们用改进的方法分离培养脂肪干细胞,并对其增殖能力和多向分化潜能进行检测。通过胰酶和胶原酶联合分次消化和换液去除红细胞,我们改进了脂肪干细胞的分离方法,可从400～600mg脂肪组织收获约5×10~5个脂肪组织来源的干细胞,并且细胞可以重叠生长一个月以上,期间细胞表现出几个对数增殖期;所有增殖的细胞其干细胞相关表面标记(CD13,CD29,CD44,CD105和CD166)都呈阳性表达;多潜能细胞相关转录因子Nanog,Oct-4,Sox-2和Rex-1也呈强阳性表达;通过油红、甲苯胺兰、ALP、von Kossa及荧光染色证明脂肪组织来源的干细胞具有向多个胚层细胞分化的能力。此外,为获得更多具有强增殖能力的细胞,根据生长曲线,我们对细胞进行每14天传代而非常规的5天传代,发现所得到的细胞仍保持强的增殖能力、干细胞表型以及更强的多向分化潜能。制备适于种子细胞生长的支架材料也是构建工程化组织的关键。在各种生物材料中,天然生物材料胶原和壳聚糖都具有较好的生物相容性和生物可降解性,但胶原的机械强度差,而壳聚糖由于其晶体结构而较坚硬,因此结合胶原和壳聚糖来制备支架将可避免两种材料的缺点,从而制备出适于种子细胞生长的支架。我们采用冻干法制备不同比例的胶原—壳聚糖支架,研究支架的孔径、孔隙率、保水性、生物可降解性以及与脂肪干细胞的生物相容性。冻干后的支架内部呈海绵状多孔隙结构,其中以胶原/壳聚糖体积比为9:1的复合支架最为疏松,1:9的支架最致密。扫描电镜下支架的胶原含量越高,支架内的胶原丝越多,支架的孔与孔之间相互连通构成了通孔。交联前后支架的形态结构无明显改变。交联后体积比为9:1,7:3和5:5的复合支架孔径为50～200μm之间,可用于细胞的三维培养。体积比为5:5的复合支架的吸水性和含水量最高,7:3和3:7次之;多孔支架在水中未发生明显的溶胀现象;支架的孔隙率均在90%以上。交联后随着胶原含量的减少,降解速率减慢,交联后的复合支架降解速度较未交联慢。脂肪干细胞在支架上培养5d后扫描电镜和HE染色可见细胞在7:3的支架上爬行生长并融合成片,而5:5支架上黏附生长的细胞较少。组织工程构建过程中,提供适于种子细胞生长的微环境也是很重要的,体外细胞生长的环境要尽量模拟体内的三维动态微环境,而转瓶生物反应器可提供三维动态微环境,促进悬浮和贴壁细胞的增殖和分化。因此我们利用转瓶生物反应器提供三维动态微环境,对支架内的脂肪干细胞进行扩增,并检测扩增后干细胞的表型特性及多向分化潜能。脂肪干细胞在转瓶生物反应器中扩增14天后,与静态条件下相比,支架内的细胞具有更强的增殖活性,扩增倍数大约为26倍,而静态条件下细胞扩增倍数近20倍;所扩增的细胞能够保持原有的干细胞表型特性和多向分化潜能。局部微环境在干细胞的定向分化中起着决定性作用,而心肌样微环境可促进骨髓干细胞向心肌细胞分化,因此心肌样微环境也可能促进脂肪干细胞向心肌细胞分化。我们分别对脂肪干细胞和心肌细胞进行间接和直接共培养研究,并检测共培养后脂肪干细胞超微结构的变化以及心肌特异性蛋白和转录因子的表达。经2周共培养后,分化的脂肪干细胞呈现心肌样超微结构,并表达心肌特异性蛋白和转录因子,流式细胞仪分析结果显示,间接共培养2周后约20%的细胞表达心肌特异性蛋白,而直接共培养2周后约30～40%的细胞表达心肌特异性蛋白,并且直接共培养体系中分化的脂肪干细胞的心肌特异性蛋白和转录因子的表达率明显高于间接共培养体系中的表达率。在各种生长因子中,人胰岛素样生长因子(IGF-1)在心脏发生和发育过程中起着重要的作用,能促进早期心肌分化。因此我们利用IGF-1基因作为目的基因,整合到支架内并转染脂肪干细胞,应用心肌细胞培养基作为诱导培养基,转瓶生物反应器提供三维动态微环境,研究脂肪干细胞在胶原-壳聚糖支架内向心肌细胞分化的情况。结果显示动态微环境能促进质粒DNA的释放和转染;IGF-1可促进脂肪干细胞在胶原-壳聚糖支架内增殖以及向心肌细胞分化;动态微环境可加强IGF-1的促进脂肪干细胞增殖分化作用。本研究采用改进的方法可较容易的获得大量脂肪干细胞,并发现脂肪干细胞具有较强的增殖能力和多向分化潜能;所制备的胶原-壳聚糖多孔支架具有较好生物相容性和生物可降解性,适于脂肪干细胞的三维培养;所设计的支架—转瓶培养系统是一个简便有效的扩增脂肪干细胞的方法;心肌样微环境可促进脂肪干细胞向心肌细胞分化;将细胞生长的支架内的阳离子多聚物作为IGF-1基因转染的载体,这种转染方法较传统的方法更简易,效率更高;联合IGF-1基因、心肌细胞培养基和动态微环境多因素刺激,可促进脂肪干细胞在支架内向心肌细胞分化。此研究对体外构建工程化心肌样组织进行心肌再生有着重要的指导意义。
[Abstract]:At present, myocardial infarction is still one of the diseases with high morbidity and mortality. Due to the limited regenerative ability of cardiac myocytes, necrotic myocardial tissue can only be replaced by non contractile scar tissue even by reperfusion therapy. Cell transplantation can only repair small area of myocardial injury, and tissue engineering technique is a large area of myocardial defect. It provides a better method of treatment. Tissue engineering includes three elements: seed cells, scaffold materials and bioactive molecules.
Stem cells, due to their self renewal and multidirectional differentiation potential, will become important tissue engineering seed cells. However, because of embryonic stem cells, bone marrow stem cells and induced pluripotent stem cells, there are ethical or immune rejection and even the risk of tumorigenesis, and fat stem cells are easily raised because of their simple and convenient sources. There is no immune rejection after transplantation, and it will become a promising seed cell in tissue engineering. Therefore, we use an improved method to isolate and culture fat stem cells and detect their proliferation and pluripotent differentiation potential. We have improved the fat dry through the combined digestion of trypsin and collagenase and the removal of red blood cells. The cell separation method can harvest about 5 * 10~5 adipose tissue derived stem cells from 400 to 600mg adipose tissue, and the cells can overlap and grow for more than a month. During the period, the cells show several logarithmic proliferation periods; all the proliferating cells have positive expression of the stem cell related surface markers (CD13, CD29, CD44, CD105 and CD166); multipotential Cell related transcription factors, Nanog, Oct-4, Sox-2 and Rex-1, are also strongly positive; the stem cells derived from adipose tissue have the ability to differentiate into multiple germ cells through oil red, methaniline, ALP, von Kossa and fluorescent staining. In addition, to obtain more cells with strong proliferative ability, we carry out the cells according to the growth curve. After 14 days of passage instead of conventional 5 days passage, it was found that the cells still maintained strong proliferative capacity, stem cell phenotype and stronger multidirectional differentiation potential.
The key to the preparation of scaffold materials suitable for seed cell growth is also the key to the construction of engineered tissue. In various biological materials, natural biomaterials collagen and chitosan have good biocompatibility and biodegradability, but the mechanical strength of collagen is poor, and chitosan is hard because of its crystal structure, so it is combined with collagen and chitosan. Sugar scaffolds will avoid the shortcomings of the two materials and prepare a scaffold suitable for seed cell growth. We use a freeze-drying method to prepare different proportions of collagen chitosan scaffolds to study the pore size, porosity, water retention, biodegradability and biocompatibility with fat stem cells. The composite scaffold with collagen / chitosan volume ratio of 9:1 was most loose and the 1:9 scaffold was the densest. The higher the collagen content of the stent under scanning electron microscope, the more collagenes in the scaffold and the interconnected pores between the scaffolds and the pores. The pore size of the composite scaffold with a ratio of 9:1,7:3 and 5:5 is 50~200 u m, which can be used for three-dimensional culture of cells. The water absorption and water content of the composite scaffold with a volume ratio of 5:5 is the highest, 7:3 and 3:7 are the second. The porous scaffold has no obvious swelling phenomenon in the water; the porosity of the scaffold is above 90%. After cross linking, the content of collagen decreases and decreases. The rate of degradation was slow and the degradation rate of the composite scaffold after crosslinking was slower than that of non crosslinking. After the 5D was cultured on the scaffold, the cells grew and fused on the 7:3 scaffold, while the cells attached to the 5:5 scaffold were less.
During the construction of tissue engineering, it is also important to provide a microenvironment suitable for the growth of seed cells. The environment in vitro cell growth is to simulate three-dimensional dynamic microenvironment in vivo, and the vase bioreactor can provide three-dimensional dynamic microenvironment to promote the proliferation and differentiation of suspended and adherent cells. Therefore, we use the bioreaction of the vase. The apparatus provides a three-dimensional dynamic microenvironment to amplify the fat stem cells in the scaffold and detect the phenotypic characteristics and pluripotent differentiation potential of the expanded stem cells. The cells in the stents have a stronger proliferation activity compared with the static conditions for 14 days after the amplification of the stem cells in a vase bioreactor, and the multiplier of the expansion is about 26 times. Under the condition, the amplification times of cells were nearly 20 times, and the expanded cells could maintain the phenotypic and pluripotent differentiation potential of stem cells.
Local microenvironment plays a decisive role in the directional differentiation of stem cells, and myocardial microenvironment can promote the differentiation of bone marrow stem cells into cardiomyocytes. Therefore, myocardial microenvironment may also promote the differentiation of adipose stem cells into cardiomyocytes. We have conducted indirect and direct co culture studies on adipose stem cells and cardiomyocytes, respectively. The ultrastructural changes of adipose stem cells and the expression of specific protein and transcription factors were observed after co culture. After 2 weeks of co culture, the differentiated adipose stem cells showed myocardial ultrastructure and expressed the specific protein and transcription factors of the myocardium. The flow cytometry showed that about 20% of the cells were expressed after 2 weeks of indirect co culture. Myocardium specific protein was expressed in 30 to 40% cells after 2 weeks of direct co culture, and the expression rate of specific protein and transcription factors of the differentiated adipose stem cells in the direct co culture system was significantly higher than that in the indirect co culture system.
In various growth factors, human insulin-like growth factor (IGF-1) plays an important role in the process of cardiac development and development, which can promote early myocardial differentiation. Therefore, we use the IGF-1 gene as the target gene to integrate into the stent and transfect the adipose stem cells, use the cardiomyocyte culture medium as the inducible medium, and turn the vase biological reaction. A three-dimensional dynamic microenvironment was provided to study the differentiation of adipose stem cells into cardiomyocytes in collagen chitosan scaffold. The results showed that dynamic microenvironment could promote the release and transfection of plasmid DNA; IGF-1 could promote the proliferation of adipose stem cells in collagen chitosan scaffold and differentiate into cardiomyocytes; dynamic microenvironment can strengthen IGF-1 It promotes the proliferation and differentiation of adipose stem cells.
This study can easily obtain a large number of fat stem cells, and found that fat stem cells have strong proliferation ability and multidirectional differentiation potential. The prepared collagen chitosan porous scaffold has good biocompatibility and biodegradability, suitable for three-dimensional culture of fat stem cells; the designed scaffold - turn bottle The culture system is a simple and effective method for amplification of fat stem cells. Myocardial microenvironment can promote the differentiation of fat stem cells into cardiomyocytes, and the cationic polypeptides in the scaffold of the cells are used as the carrier of IGF-1 gene transfection. The transfection method is simpler and more efficient than the traditional method. Combined with the IGF-1 gene, the myocardium is fine. The multi factor stimulation of cell culture medium and dynamic microenvironment can promote the differentiation of adipose stem cells into cardiomyocytes in the scaffold. This study has important guiding significance for the construction of myocardium like tissue in vitro for construction of engineered myocardium.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329;R542.22

【引证文献】