蚕丝人工韧带复合骨髓基质干细胞的实验研究
发布时间：2018-11-17 14:46
【摘要】：目的 利用自制的蚕丝人工韧带支架，多种方法对表面结构进行改性，观察对比不同分组间种子细胞粘附、生长情况，为组织工程化人工韧带支架材料与种子细胞复合提供一种理想的方法。 方法 蚕丝人工韧带支架材料，采用胰酶、碳酸钠联合脱胶，称重测韧带脱胶率，以EDC（1-乙基-3-(3-二甲基氨丙基)碳二亚胺）/NHS（N-羟基琥珀酰亚胺）作为交联剂进行胶原的交联，酶标仪法测其交联度。采用荧光显微镜、扫描电镜观察蚕丝脱胶及与交联胶原复合后的表面结构变化。 把韧带支架分为试验组、参照组和空白对照组。参照组蚕丝人工韧带与胶原结合形成蚕丝-胶原结合韧带支架，不做交联处理；试验组采用EDC/NHS对蚕丝-胶原结合韧带支架材料进行交联；空白组未作任何处理。兔的骨髓基质干细胞（Marrowmesenchymal stem cells，MSCs）全血贴壁法培养至第八代，通过CD34、CD44、CD50免疫组化及流式细胞仪进行干细胞检测，调整细胞密度为1×106/L，分别种植于三种支架材料上，对三种材料表面培养的细胞进行了荧光显微镜、扫描电镜及酶标仪的检测。比较不同分组间细胞粘附、增殖生长情况。 结果 采用胰酶、碳酸钠联合脱胶，能将丝胶基本去除，脱胶率29%左右。当EDC浓度为3g/L时，交联度最大。荧光显微镜下未脱胶蚕丝表面比较完整；脱胶蚕丝结构变得蓬松，表面光滑，丝素纤维暴露。SEM观察，未脱胶蚕丝表面粗糙不平，丝胶包绕在丝素纤维周围；脱胶完全的蚕丝表面很光滑，可见单根的丝素纤维。荧光倒置显微镜及SEM下复合胶原后的韧带可见，胶原呈一薄层分布在韧带表面，呈不规则的线状突起，色泽较白。交联后胶原-蚕丝韧带成蜂窝状。 全血贴壁法体外培养的BMSC，检测CD34阴性、CD44、CD50、STRO-1阳性，流式细胞仪检测CD34+细胞占10%左右；CD44+占42%左右，表现为干细胞特性。荧光显微镜下，未交联胶原-蚕丝韧带细胞数目较少，细胞未见舒展；交联后的胶原-蚕丝人工韧带，蚕丝表面粗糙，细胞粘附率较高，舒展充分，蚕丝之间的间隙模糊。SEM下见，交联后的蚕丝人工韧带与细胞贴合紧密，细胞伸展充分，有较多的触角伸出，明显优于其它组。经单因素方差分析，试验组与参照组、空白对照组比较P＜0.05，差异有统计学意义。 结论 1.骨髓基质干细胞能够向多种组织细胞分化，在实验中表现出良好的增殖能力，是种子细胞的理想选择。 2.胶原蛋白具有良好的生物相容性，是一种良好的蚕丝表面修饰剂。EDC毒性很低，可作为良好的交联剂。 3.经过EDC/NHS交联的蚕丝-胶原结合韧带支架材料，能够显著提高细胞黏附率。 意义 随着人们物质文化水平的不断提高和体育事业的蓬勃发展，韧带损伤病例呈现快速增长的趋势。韧带损伤后，往往需要韧带重建手术治疗。目前韧带移植材料有①自体韧带；②异体韧带；③人工韧带，都存在来源少，免疫排斥及传播疾病的缺陷。组织工程人工韧带的发展，为治疗韧带损伤提供了另一条途径。骨髓间充质干细胞能够向多种组织细胞分化，是组织工程人工韧带理想的种子细胞。蚕丝具备良好的生物相容性和细胞亲和力，可作为良好的组织工程支架材料，由于材料表面过于光滑、致密，，种子细胞不能与蚕丝韧带复合。本实验通过对蚕丝韧带进行改性，克服了这一难题，为组织工程化人工韧带的发展提供有力的实验支持。
[Abstract]:Purpose The surface structure was modified by using a self-made artificial ligament support, and the adhesion and growth of the seed cells in different groups were observed. The method The method for preparing the silk artificial ligament support material adopts the combination of the pancreatin and the sodium carbonate for degumming, and the degumming rate of the ligament is measured, and the EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/ NHS (N-hydroxy-amber-imine) is used as the crosslinking agent. the crosslinking of the collagen, the enzyme, The cross-linking degree of the cross-linked collagen was measured by means of a fluorescence microscope and a scanning electron microscope. The cross-linking collagen was combined with the cross-linked collagen. The posterior surface structure changes. The ligamentum support is divided into two groups. In group, reference group and blank control group, the silk-collagen combined ligament stent was formed with the combination of the artificial ligament of the reference group and the collagen, and the cross-linking treatment was not performed; the test group used the EDC/ NHS to carry out the cross-linking of the silk-collagen-binding ligament support material. The bone marrow stromal cells (MSCs) of the rabbit were cultured to the eighth generation by means of the whole blood-adhering method of the bone marrow stromal cells (MSCs), and the stem cells were detected by CD34, CD44, CD50 immunohistochemistry and flow cytometry, and the density of the cells was 1-106/ L. the three kinds of support materials are not planted, and the cells cultured on the surface of the three materials are used for fluorescence microscopy, The detection of the scanning electron microscope and the microplate reader. The comparison of the different groups cell The result is that pancreatin and sodium carbonate are used for degumming, can basically remove the sericin, and the degumming rate is about 29%. The cross-linking degree is the largest when the concentration of EDC is 3g/ L. The surface of the unglued silk under the fluorescence microscope is relatively complete; the degummed silk knot The structure becomes fluffy, the surface is smooth, and the silk fibroin fiber is exposed. The SEM observation shows that the surface of the non-degummed silk is not rough, the silk glue bag is wound around the silk fibroin fiber, and the degumming is complete. The surface of the silk is smooth, and a single silk fibroin fiber can be seen. The fluorescence inversion microscope and the ligament of the composite collagen under the SEM are visible, and the collagen is distributed in a thin layer in the ligament. a surface, an irregular linear protrusion, a color, After cross-linking, the collagen-silk ligament was made into a honeycomb shape. The BMSC, which was cultured in vitro by the whole blood-attached method, was used to detect CD34 +, CD44, CD50 and STRO-1, and the CD34 + cells were detected by flow cytometry. CD44 + accounted for about 42%, and expressed as a stem cell characteristic. Under the fluorescence microscope, the number of uncrosslinked collagen-silk ligament cells is small and the cells are not stretched; the cross-linked collagen-silk artificial ligament, the surface of the silk, the cells, the adhesion rate is high, the stretching is sufficient, the gap between the silk is blurred, The show is full, with more tentacles extending, which is obviously superior to other groups. The single-factor analysis of variance, the test group and the reference group, the blank control Conclusion 1. The bone marrow stromal stem cells can differentiate into many kinds of tissue cells. and it is an ideal choice for seed cells. The collagen has good biocompatibility, A good surface modification agent for silk. The toxicity of EDC is very low, which can be used as a good cross-linking agent. 3. EDC /N HS-crosslinked silk-collagen combined with the ligament stent material can significantly improve the cell adhesion rate. The continuous improvement of the level of material culture and the construction of the sports cause In the development of the ligament, there is a trend of rapid growth in the case of ligament damage. The ligament is often used for the reconstruction of the ligament after the ligament is damaged. The presence of an autograft of the ligamentum; an allogenic ligament; an artificial ligament, both of which are of low origin, immune, The development of artificial ligament of tissue engineering provides another way for the treatment of ligament damage. The bone marrow mesenchymal stem cells can be differentiated into a plurality of tissue cells and are the ideal seed cells for tissue engineering artificial ligaments. The silk has good biocompatibility and cell affinity, and can be used as a good The tissue engineering scaffold material has the advantages that the surface of the material is too smooth and dense, and the seed cell cannot be compounded with the silk ligament.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2
本文编号：2338162
[Abstract]:Purpose The surface structure was modified by using a self-made artificial ligament support, and the adhesion and growth of the seed cells in different groups were observed. The method The method for preparing the silk artificial ligament support material adopts the combination of the pancreatin and the sodium carbonate for degumming, and the degumming rate of the ligament is measured, and the EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/ NHS (N-hydroxy-amber-imine) is used as the crosslinking agent. the crosslinking of the collagen, the enzyme, The cross-linking degree of the cross-linked collagen was measured by means of a fluorescence microscope and a scanning electron microscope. The cross-linking collagen was combined with the cross-linked collagen. The posterior surface structure changes. The ligamentum support is divided into two groups. In group, reference group and blank control group, the silk-collagen combined ligament stent was formed with the combination of the artificial ligament of the reference group and the collagen, and the cross-linking treatment was not performed; the test group used the EDC/ NHS to carry out the cross-linking of the silk-collagen-binding ligament support material. The bone marrow stromal cells (MSCs) of the rabbit were cultured to the eighth generation by means of the whole blood-adhering method of the bone marrow stromal cells (MSCs), and the stem cells were detected by CD34, CD44, CD50 immunohistochemistry and flow cytometry, and the density of the cells was 1-106/ L. the three kinds of support materials are not planted, and the cells cultured on the surface of the three materials are used for fluorescence microscopy, The detection of the scanning electron microscope and the microplate reader. The comparison of the different groups cell The result is that pancreatin and sodium carbonate are used for degumming, can basically remove the sericin, and the degumming rate is about 29%. The cross-linking degree is the largest when the concentration of EDC is 3g/ L. The surface of the unglued silk under the fluorescence microscope is relatively complete; the degummed silk knot The structure becomes fluffy, the surface is smooth, and the silk fibroin fiber is exposed. The SEM observation shows that the surface of the non-degummed silk is not rough, the silk glue bag is wound around the silk fibroin fiber, and the degumming is complete. The surface of the silk is smooth, and a single silk fibroin fiber can be seen. The fluorescence inversion microscope and the ligament of the composite collagen under the SEM are visible, and the collagen is distributed in a thin layer in the ligament. a surface, an irregular linear protrusion, a color, After cross-linking, the collagen-silk ligament was made into a honeycomb shape. The BMSC, which was cultured in vitro by the whole blood-attached method, was used to detect CD34 +, CD44, CD50 and STRO-1, and the CD34 + cells were detected by flow cytometry. CD44 + accounted for about 42%, and expressed as a stem cell characteristic. Under the fluorescence microscope, the number of uncrosslinked collagen-silk ligament cells is small and the cells are not stretched; the cross-linked collagen-silk artificial ligament, the surface of the silk, the cells, the adhesion rate is high, the stretching is sufficient, the gap between the silk is blurred, The show is full, with more tentacles extending, which is obviously superior to other groups. The single-factor analysis of variance, the test group and the reference group, the blank control Conclusion 1. The bone marrow stromal stem cells can differentiate into many kinds of tissue cells. and it is an ideal choice for seed cells. The collagen has good biocompatibility, A good surface modification agent for silk. The toxicity of EDC is very low, which can be used as a good cross-linking agent. 3. EDC /N HS-crosslinked silk-collagen combined with the ligament stent material can significantly improve the cell adhesion rate. The continuous improvement of the level of material culture and the construction of the sports cause In the development of the ligament, there is a trend of rapid growth in the case of ligament damage. The ligament is often used for the reconstruction of the ligament after the ligament is damaged. The presence of an autograft of the ligamentum; an allogenic ligament; an artificial ligament, both of which are of low origin, immune, The development of artificial ligament of tissue engineering provides another way for the treatment of ligament damage. The bone marrow mesenchymal stem cells can be differentiated into a plurality of tissue cells and are the ideal seed cells for tissue engineering artificial ligaments. The silk has good biocompatibility and cell affinity, and can be used as a good The tissue engineering scaffold material has the advantages that the surface of the material is too smooth and dense, and the seed cell cannot be compounded with the silk ligament.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2
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