应用脂肪干细胞与丝素蛋白和壳聚糖支架材料共同构建细胞—支架复合物的实验研究
发布时间：2018-07-25 07:58
【摘要】：目的: 研究脂肪干细胞向软骨细胞培养诱导的方法;用丝素蛋白和壳聚糖作为三维多孔支架材料,研究它的制作方法和特点;探讨用诱导后的软骨细胞与丝素蛋白和壳聚糖支架材料体外构建细胞-支架复合物的可行性。 方法: (1)采用贴壁分离方法从人体脂肪组织抽吸液中分离出脂肪干细胞,培养扩增,传到第三代时在骨形态发生蛋白和碱性成纤维细胞生长因子的软骨细胞诱导培养基中,诱导脂肪干细胞向软骨细胞转化。Ⅱ型胶原免疫组织化学检测细胞特性。 (2)以丝素蛋白、壳聚糖为支架原料,蚕丝经过溶解、脱胶、透析后得丝素蛋白溶液,与壳聚糖溶液混合溶解,得混合溶液。冷冻干燥法制成丝素蛋白/壳聚糖三维立体多孔支架材料,通过体积法计算支架孔隙率,通过扫描电镜对支架的外观和性能进行观察和测量。 (3)将诱导后的细胞接种于消毒灭菌过的支架材料上,细胞与支架复合物联合培养。扫描电镜观察细胞-支架复合物的外观和内部结构,以及细胞在支架里的生长情况。 结果: (1)脂肪组织通过消化得到脂肪干细胞,并且通过培养基培养长势良好。三代的脂肪干细胞在骨形态发生蛋白和碱性成纤维细胞生长因子的软骨诱导培养基中诱导培养,细胞的形态发生改变,呈软骨细胞的特点。由长梭形变为多边形,Ⅱ型胶原免疫组织化学染色呈阳性。 (2)制作完成的丝素蛋白/壳聚糖三维多孔支架呈白色,呈海绵状,可见纤维状排列。支架材料表面均匀,柔软有弹性,孔隙大小均匀,没有明显突起和凹陷。支架的孔隙率大于80%。以不同比例制作的支架以丝素蛋白和壳聚糖的比例5:5和4:6最为合适。 (3)诱导后的细胞接种在支架上在培养基中联合培养,扫描电镜显示细胞与支架黏附良好,细胞可以在支架材料里面贴附生长。 结论: (1)脂肪干细胞在软骨培养基中,通常单独应用一种细胞生长因子都可以诱导其成软骨细胞。在本实验中联合应用骨形态发生蛋白14和碱性成纤维细胞生长因子两种细胞生长因子较单独应用骨形态发生蛋白能更好的诱导脂肪干细胞向软骨细胞方向分化。细胞长的更好,更密集。 (2)蚕丝和壳聚糖两种天然材料通过混合溶解得到的多孔三维立体支架材料经测得空隙率大于80%,有利于细胞的渗透和营养物质的交换。三维立体环境也可以给细胞提供适宜的微环境。所有丝素蛋白/壳聚糖支架可以作为软骨组织工程的三维多孔支架。 (3)脂肪干细胞经过诱导后的软骨细胞可以很好的附着在丝素蛋白和壳聚糖支架上,细胞可以在表面和内部生长。本实验的细胞-支架复合物可以用来作为组织工程软骨的实验研究。
[Abstract]:Objective:
The methods for inducing the induction of adipose stem cells to chondrocytes were studied. The methods and characteristics were studied by using silk fibroin and chitosan as a three-dimensional porous scaffold material. The feasibility of constructing the cell scaffold complex in vitro with fibroin and chitosan scaffold materials was discussed.
Method:
(1) the fat stem cells were isolated from the sucking fluid of human adipose tissue by the method of adherent wall separation, and were cultured and amplified to be transmitted to the third generation of chondrocytes induced by bone morphogenetic protein and basic fibroblast growth factor to induce the transformation of fat stem cells to chondrocytes. Sex.
(2) the silk fibroin and chitosan were used as the scaffold. The silk fibroin protein solution was dissolved, degumbed and dialysated, and the chitosan solution was mixed and dissolved. The mixed solution was obtained. The freeze-drying method was used to calculate the three-dimensional porous scaffold of fibroin protein / chitosan. The scaffold porosity was calculated by the volume method. The appearance and appearance of the scaffold were examined by scanning electron microscope. The performance is observed and measured.
(3) the induced cells were inoculated on the sterilized scaffold material, and the cells were co cultured with the scaffold complex. The appearance and internal structure of the cell scaffold complex and the growth of the cells in the scaffold were observed by scanning electron microscope.
Result:
(1) adipose tissue is digested to obtain fat stem cells, and the growth potential is good by culture medium. The three generation of fat stem cells are induced in the cartilage induced medium of bone morphogenetic protein and basic fibroblast growth factor, and the morphological changes of the cells are characterized by the characteristics of chondrocytes. The long spindle is a polygon, II. The immuno histochemical staining of type collagen was positive.
(2) the three-dimensional porous scaffolds made of silk fibroin / chitosan were white, spongy and fibrous. The surface of the scaffold was uniform, soft and elastic, the size of the pores was uniform, no obvious protruding and depression. The ratio of scaffolds with different proportions of 80%. and 5:5 and 4:6 was the best ratio of silk fibroin and chitosan. It's right.
(3) the induced cell inoculation was co cultured on the scaffold in the culture medium. The scanning electron microscope showed that the cells and the scaffold adhered well, and the cells could attach the growth in the scaffold material.
Conclusion:
(1) in the cartilage medium, adipose stem cells usually use a single cell growth factor to induce their chondrocytes. In this experiment, the combination of bone morphogenetic protein 14 and basic fibroblast growth factor two cell growth factors can better induce adipose stem cells than the single use of bone morphogenetic protein. Chondrocytes differentiate in a more compact way.
(2) the porous three-dimensional scaffold material obtained by mixed solution of two kinds of silk and chitosan can be obtained through mixed dissolution of the porous three-dimensional scaffold material, the void ratio is greater than 80%, which is beneficial to the infiltration of cells and the exchange of nutrients. The three-dimensional environment can also provide the suitable microenvironment for the cells. All silk egg white / chitosan scaffolds can be used as cartilage tissue engineering. The three-dimensional porous scaffold.
(3) the chondrocytes of the adipose stem cells can be well attached to the silk fibroin and chitosan scaffold after the induction of the stem cells. The cells can grow on the surface and inside. The cell scaffold complex in this experiment can be used as an experimental study of tissue engineered cartilage.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2143153
[Abstract]:Objective:
The methods for inducing the induction of adipose stem cells to chondrocytes were studied. The methods and characteristics were studied by using silk fibroin and chitosan as a three-dimensional porous scaffold material. The feasibility of constructing the cell scaffold complex in vitro with fibroin and chitosan scaffold materials was discussed.
Method:
(1) the fat stem cells were isolated from the sucking fluid of human adipose tissue by the method of adherent wall separation, and were cultured and amplified to be transmitted to the third generation of chondrocytes induced by bone morphogenetic protein and basic fibroblast growth factor to induce the transformation of fat stem cells to chondrocytes. Sex.
(2) the silk fibroin and chitosan were used as the scaffold. The silk fibroin protein solution was dissolved, degumbed and dialysated, and the chitosan solution was mixed and dissolved. The mixed solution was obtained. The freeze-drying method was used to calculate the three-dimensional porous scaffold of fibroin protein / chitosan. The scaffold porosity was calculated by the volume method. The appearance and appearance of the scaffold were examined by scanning electron microscope. The performance is observed and measured.
(3) the induced cells were inoculated on the sterilized scaffold material, and the cells were co cultured with the scaffold complex. The appearance and internal structure of the cell scaffold complex and the growth of the cells in the scaffold were observed by scanning electron microscope.
Result:
(1) adipose tissue is digested to obtain fat stem cells, and the growth potential is good by culture medium. The three generation of fat stem cells are induced in the cartilage induced medium of bone morphogenetic protein and basic fibroblast growth factor, and the morphological changes of the cells are characterized by the characteristics of chondrocytes. The long spindle is a polygon, II. The immuno histochemical staining of type collagen was positive.
(2) the three-dimensional porous scaffolds made of silk fibroin / chitosan were white, spongy and fibrous. The surface of the scaffold was uniform, soft and elastic, the size of the pores was uniform, no obvious protruding and depression. The ratio of scaffolds with different proportions of 80%. and 5:5 and 4:6 was the best ratio of silk fibroin and chitosan. It's right.
(3) the induced cell inoculation was co cultured on the scaffold in the culture medium. The scanning electron microscope showed that the cells and the scaffold adhered well, and the cells could attach the growth in the scaffold material.
Conclusion:
(1) in the cartilage medium, adipose stem cells usually use a single cell growth factor to induce their chondrocytes. In this experiment, the combination of bone morphogenetic protein 14 and basic fibroblast growth factor two cell growth factors can better induce adipose stem cells than the single use of bone morphogenetic protein. Chondrocytes differentiate in a more compact way.
(2) the porous three-dimensional scaffold material obtained by mixed solution of two kinds of silk and chitosan can be obtained through mixed dissolution of the porous three-dimensional scaffold material, the void ratio is greater than 80%, which is beneficial to the infiltration of cells and the exchange of nutrients. The three-dimensional environment can also provide the suitable microenvironment for the cells. All silk egg white / chitosan scaffolds can be used as cartilage tissue engineering. The three-dimensional porous scaffold.
(3) the chondrocytes of the adipose stem cells can be well attached to the silk fibroin and chitosan scaffold after the induction of the stem cells. The cells can grow on the surface and inside. The cell scaffold complex in this experiment can be used as an experimental study of tissue engineered cartilage.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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