标题: 以基质金属蛋白酶建立具细胞专一生物可降解性之超分子水胶应用于三维人类骨髓间叶干细胞培养
Matrix Metalloproteinases-mediated Cell-type Specific Biodegradable Supramolecular Hydrogel for Three-dimensional Human Bone Marrow Mesenchymal Stem Cells Cultivation
作者: 杨孟融
Yang, Meng-Jung
柯富祥
Ko, Fu-Hsiang
材料科学与工程学系奈米科技硕博士班
关键字: NapFF;超分子水胶;人类骨髓间叶干细胞;三维培养;基质金属蛋白酶;NapFF;Supramolecular hydrogels;Human bone marrow mesenchymal stem cells;Three-dimensional cultivation;Matrix metalloproteinase
公开日期: 2012
摘要: Naphthalene -Phe-Phe (NapFF)为生物化学中常用的超分子序列,常被作为药物释放、细胞内调控和酵素活性分析的工具。许多文献指出,NapFF不仅可以作为自组装过程中,有效的成胶组件,也具有高度生物相容性和酵素调控成胶之特性,可应用在组织工程、再生医学和注射治疗等领域。然而,目前生物相容性试验中,只利用低浓度之NapFF进行平面液态培养,尚未达到成胶浓度,以应用在三维组织工程研究上。因此,我们分别以海拉细胞和人类骨髓间叶干细胞(Human bone marrow mesenchymal stem cells; hMSCs)为模型,进行NapFF水胶的三维细胞培养,利用DNA含量和明胶蛋白酵素电泳法,测定细胞增殖率、细胞存活度及进行特定蛋白质分析。
目前,我们加入含有氯化钙之细胞悬浮液,已成功的建立NapFF三维之含细胞的超分子水胶。利用FTIR、CD spectra和SEM images,厘清钙离子和NapFF上之羧基和酰胺的交互作用,并如何诱发NapFF进行自组装并成胶的分子机转,并影响到NapFF之奈米纤维尺度大小。此外,在细胞培养的实验中,发现hMSCs在NapFF超分子水胶中,相对海拉细胞有较好的细胞增殖率和细胞存活度,且会降解NapFF水胶。在明胶蛋白酵素电泳法分析中,hMSCs的基质金属蛋白酶(Matrix metalloproteinase-2; MMP-2)活性相对海拉细胞具有显着性的差异。因此我们认为不同细胞之MMPs的活性差异,会导致细胞增殖与否和水胶的降解,而实验显示NapFF对于hMSCs具有细胞培养专一性。
为了进一步证明NapFF超分子水胶可应用在三维列印技术的可能性,我们包埋萤光分子代替生长因子在钙离子诱发之NapFF自组装水胶,测量萤光分子之扩散速率。研究结果显示,含有氯化钙的培养液,可以有效的抑制萤光分子的扩散,表示钙离子诱发之NapFF自组装水胶可以包埋生长因子,建立良好的细胞微环境提供干细胞增殖并分化。
NapFF is a small unique unnatural amino acid. It has a potential to act as a small molecule hydrogelator. This may leads to the formation of NapFF supramolecular hydrogels, a unique type of soft material that consist of water. Apart, from NapFF’s ability to form building blocks for supramolecular self-assembly it has also excellent biocompatibility. Owing, to these properties NapFF have attracted much attention in biochemistry for its application in drug release, inner cellular regulation and enzyme activity analysis. The above mentioned properties of NapFF can further be exploited for its application in tissue engineering, regenerative medicine and minimal invasive threapy. However, before its practical application in these fields there are number of challenges need to be met such as low concentration of cultivated cells with NapFF and liquid form in biocompatibility test, less than the gelation concentrations for three-dimensional (3D) tissue engineering. In this study, we have successfully encapsulated HeLa cells and human bone marrow mesenchymal stem cells (hBMSCs) in 3D supramolecular hydrogel of NapFF in vitro. Quantifying DNA contents and enzyme activities were evaluated by studying the cells proliferation rates, viability and protein expressions.
In this study, 3D cells-encapsulating supramolecular hydrogel of NapFF has been fabricated successfully via cell suspensions containing CaCl2 supplement. The FTIR, CD spectra and SEM study, is applied successfully to study the interactions of Ca2+ with the carboxylic acid and amide groups of NapFF. This type of metal ion interaction, also contributes to the self-assembly of hydrogels which further affected the diameter of NapFF nanofibers. In the cell encapsulation experiments, hBMSCs showed better proliferation rate, viability and biodegradability in NapFF supramolecular hydrogel, as compared with HeLa cells. According to gelatin zymography, the matrix metalloproteinase-2 (MMP-2) activity of hBMSCs was approximately 10-fold higher than HeLa cells. We proposed that the difference between enzyme activities in cells have permitted the cell proliferation and NapFF degradation. All of these results indicate that NapFF equipped with the characteristic of cell-type specific 3D cultivation for hBMSCs.
Further, to apply this study in 3D printing technique a NapFF supramolecular hydrogel, with a fluorescence molecule embedded on it was prepared. This was done to substitute growth factor in Ca2+-induced NapFF hydrogels, and the diffusion ability is studied. On the basis of these experiments, it is concluded that the diffusion of fluorescence molecule can be suppressed by medium containing CaCl2. The study can also be applied to establish cellular microenvironment, which will be suitable for stem cells proliferation and differentiation.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT070051613
http://hdl.handle.net/11536/71971
显示于类别:Thesis