【作者】 谢炜；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2010， 硕士

【摘要】 丝素蛋白是一种天然合成的高分子聚合物,作为手术缝线用于临床已有一百多年的历史。近年来,由于其低免疫原性,良好的生物相容性,缓慢的降解速度及特有的理化性质,丝素蛋白作为一种新的生物材料应用越来越受到欢迎。柞蚕丝素是除家蚕丝素外的另一大主要品种,在氨基酸组成上柞蚕丝素和家蚕丝素都主要由甘氨酸和丙氨酸两种氨基酸组成,但柞蚕丝素中丙氨酸的含量比家蚕丝素要高,且含有丰富的精氨酸-甘氨酸-天门冬氨酸序列(RGD序列),更有利于细胞的粘附和生长。间充质干细胞(MSC)是干细胞的一种,是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中,因能分化为间质组织而得名,具有亚全能分化潜能。间充质干细胞具有干细胞的共性:即自我更新,多向分化和归巢的能力。在特定的环境下,能够分化为多种组织细胞。而且易于富集和扩增,不涉及伦理问题,在细胞治疗、组织器官修复、发育生物学、药物等领域有着极为广阔的应用前景,已被越来越广泛的应用到组织工程中,是理想的种子细胞。随着医学生物研究地深入,细胞与基质材料的相互作用已成为组织工程中关注的焦点。本实验采用小鼠间充质干细胞株(C3H10T1/2)和緑色荧光蛋白转基因小鼠的羊水干细胞( GFP—mAFS)这两种小鼠间充质干细胞为研究对象,探讨再生柞蚕丝素蛋白对这两种干细胞体外分化发育的支持作用。第一部分再生柞蚕丝素蛋白对小鼠间充质干细胞株体外生长和分化的研究目的探讨再生柞蚕丝素蛋白对小鼠间充质干细胞株(C3H10T1/2)体外生长和分化的支持作用,为再生柞蚕丝素蛋白应用于组织工程学研究提供理论依据。方法采用再生家蚕丝素蛋白、再生柞蚕丝素蛋白、I型胶原、普通细胞培养板为研究对象,观察小鼠间充质干细胞株(C3H10T1/2)在这四种生物材料上的粘附、生长分化以及表面抗原的变化。利用光学显微镜观察细胞生长形态;MTT法检测细胞的增殖度;流式细胞仪测定细胞表型;诱导小鼠间充质干细胞(C3H10T1/2)向成脂肪细胞分化。结果再生柞蚕丝素蛋白对于细胞生长、形态,细胞表面抗原表达均无影响。MTT法检测结果显示在培养第6天C3H10T1/2细胞在再生柞蚕丝素膜上的增殖明显(P<0.05)。C3H10T1/2细胞能向成脂肪细胞分化。结论本研究结果显示再生柞蚕丝素蛋白在体外支持C3H10T1/2细胞的粘附,生长和分化,具有很好的细胞相容性。第二部分再生柞蚕丝素蛋白对小鼠羊水干细胞体外生长的研究目的探讨再生柞蚕丝素蛋白对小鼠羊水干细胞体外生长的支持作用,为再生柞蚕丝素蛋白应用于组织工程学研究提供理论依据。方法采用再生柞蚕丝素蛋白和普通细胞培养板为研究对象,观察小鼠羊水干细胞( GFP—mAFS)在这两种生物材料上的粘附、生长情况。利用倒置相差显微镜和倒置荧光显微镜观察细胞的生长情况。结果再生柞蚕丝素蛋白对细胞的粘附和生长形态均无影响。结论本研究结果显示再生柞蚕丝素蛋白在体外支持GFP—mAFS细胞的粘附,生长,具有很好的细胞相容性。更多还原

【Abstract】 Silk fibroin is a kind of natural macromolecules, and is used as the surgical sutures with more than 100-year history. Recently, Silk fibroin is applied as a new type of biomaterial more and more broadly because of its unique characteristics including low immunogenicity, good histocompatibility, and slow rate of biodegradation and so on. Except for silk fibroin of domestic silkworm, silk fibroin of antheraea penyi is an important member of silk fibroin. Similarly to silk fibroin of domestic silkworm, silk fibroin of antheraea penyi is mainly composed of glycine and alanine. But silk fibroin of antheraea penyi is better than silk fibroin of domestic silkworm in supporting cell adherence and growth because it contains more alanine and is rich in arginine- glycine- asparticacid (RGD sequence). Mesenchymal stem cells ( MSCs) are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. These cells are uncommitted precursor cells with homing ability, and have extensive renewal potential. MSCs are the idea seeds for tissue engineering because these cells are easily to obtain and enrich, and are not involved in ethical problems. Researchers are focused on the interaction of seed cells and matrix material in tissue engineering and its mechanism. The adherent ability is one of the critical characteristics of a biomaterial matrix because the applicable biomaterial should have an idea ability to adhere to seed cells. In this study, to test the supporting ability of regenerated silk fibroin of antheraea penyi (A penyi SF) to stem cells, we studied the growth and differentiation of murine MSCs cell line C3H10T1/2 and amniotic fluid stem cells (mAFS) that cultured onA penyi SF in vitro. PartⅠThe Role of the Regenerated Antheraea Penyi Silk Fibroin in the Proliferation and Differentiation of Mouse Mesenchymal Stem Cell-line in VitroObjective In order to provide the experimental evidence for applying the regenerated antheraea penyi silk fibroin to tissue engineering, we observe the proliferation and differentiation of the mouse mesenchymal stem cell line(C3H10T1/2) on the regenerated antheraea penyi silk fibroin membrane in vitro.Methods Four biomaterials, including the regenerated silk fibroin of domestic silkworm (B mori SF), the regenerated silk fibroin of antheraea penyi (A penyi SF), collagen and the cell culture plastic, were used in the study. The ES cell line C3H10T1/2 cells that were cultured on the four biomaterials, and their adhesion ability, proliferation, differentiation and the phenotypes of were investigated by the observing under inverted microscope, immunostaining and FACS, and MTT assay.Result There is no difference of morphology and phenotype of C3H10T1/2 cells after cultured on these four different materials. The MTT assay showed that C3H10T1/2 cells proliferated more rapidly on the antheraea penyi material than that on the other 3 biomaterials after sixth day’s culture(P<0.05). Furthermore, C3H10T1/2 cells can be successfully induced and differentiated into adipocytes. Conclusion These results show that the regenerated silk fibroin of antheraea penyi provide an suitable support for the growth and differentiation of C3H10T1/2 cell, and exhibit good cellular compatibilities.Part II The Role of the Regenerated Antheraea Penyi Silk Fibroin in the Growth of Mouse Amniotic Fluid Stem Cells in VitroObjective To provide the experimental evidence for applying the regenerated antheraea penyi silk fibroin to tissue engineering, we discussed the growth of the mouse amniotic fluid stem cells from GFP mice(GFP-mAFS) on the regenerated antheraea penyi silk fibroin membrane in vitro. Methods The regenerated silk fibroin of domestic silkworm (B mori SF) and the regenerated silk fibroin of antheraea penyi (A penyi SF) were used in the study. The GFP-mAFS cells were cultured on both biomaterials, and their adhesion ability and growth were investigated by the observing under inverted microscope.Result There is no influence of difference of regenerated silk fibroin of antheraea penyi on the morphology and adherent ability of GFP-mAFS cells.Conclusion These results show that the regenerated silk fibroin of antheraea penyi provide an good support for the growth and adhrence of mAFS, and exhibit good cellular compatibilities.更多还原