RGD序列肽修饰的丝素蛋白仿生支架材料对骨髓间充质干细胞黏附、增殖的影响

Adhesion and proliferation of marrow mesenchymal stem cells cultured on silk fibroin biomaterial modified with RGD peptide

目的:为了促进种子细胞在韧带组织工程支架材料上的黏附增殖,在丝素薄膜上共价接枝人工合成的具有生物活性的GRGDS序列,并观察RGD多肽修饰的丝素蛋白支架材料对骨髓间充质干细胞的黏附、增殖的影响。方法:实验于2006-07/2007-07在华中科技大学同济医学院协和医院中心实验室和骨科实验室完成。利用市购的白色家蚕蚕丝制备丝素蛋白薄膜,在丝素蛋白支架材料表面接上海吉尔公司合成的RGD多肽,为丝素-RGD组,以未接多肽的丝素蛋白材料和细胞培养板分别作丝素组和阳性对照组。取第3代骨髓间充质干细胞接种到以上材料和培养板上培养4,12 h后,沉淀法定量检测黏附的细胞数检测细胞黏附率;培养1,2,3,4 d后比较材料上的细胞标准化细胞密度来反映细胞的增殖程度;培养24 h后对细胞骨架作免疫荧光染色,在激光共聚焦显微镜下观察两组细胞骨架的组织情况。结果:培养4,12 h时丝素-RGD组细胞黏附率均显著高于丝素组(P<0.01),但与阳性对照组无明显差异(P>0.05)。培养1,2,3,4 d时各组细胞的增殖程度无显著性差异(P>0.05)。培养24 h丝素-RGD组细胞完全铺展开,有粗大的细胞骨架组织,细胞内应力纤维清晰可见,其肌动蛋白的荧光强度显著高于丝素组(P<0.05)。结论:RGD多肽修饰能显著促进骨髓基质细胞在丝素蛋白支架材料上的黏附、铺展,但对细胞增殖无明显促进作用。

AIM： In order to promote the adhesion and proliferation of seed cell in the scaffold materials of ligament tissue engineering, the bioactive GRGDS sequence was synthesized and covalent on the silk film, and the proliferation and adhesion of marrow mesenchymal stem cell （MSC） on the modified scaffold materials with RGD peptide were investigated. METHODS： From July 2006 to July 2007, the experiment was carried out in the Central Laboratory and Orthopedic Laboratory, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. The silk film was prepared on white silkworm, and RGD peptide was modified on the surface of silk materials, serving as silk-RGD peptide group. While the silk film without modification and culture plate ware taken as silk group and positive control group, respectively. The third generation of MSC was seeded onto the RGD modified silk film, the silk film and the culture plate, and then incubated for 4 hours and 12 hours. In each experiment, the number of adhesive cells was counted by using precipitation method to evaluate the efficiency of cell adhesion; At days 1, 2, 3 and 4 of inoculation, the cell density was calculated and used as index of cell proliferation. MSC was seeded onto the RGD modified silk film and the silk film for 24 hours, the immunofluorescence technique was adopted to label the cell skeleton, and the skeletal tissues ware observed with the Laser Confocal Microscopy. RESULTS： After 4 hours and 12 hours culture, the efficiency of cell adhesion in the modified silk scaffold was significantly higher than that on unmodified silk scaffold （P 〈 0.01）, but no significant difference was found between the modified silk scaffold and the culture plate （P 〉 0.05）. There were no significant differences in cellular proliferation among each group for culture of 1 day, 2 days, 3 days and 4 days （P 〉 0.05）. After 24-hour culture, the cells on the modified silk film spread widely, their cytoskeleton was more robust, and intracellular stress fiber appeared evidently. The fluorescence intensity of actin on the modified silk film was significantly higher than that on the unmodified silk film （P 〈 0.05）. CONCLUSION： RGD peptide modification can enhance the adhesion and spread of MSC to silk scaffold, but no obvious effects are observed in cell proliferation.