负载纳米银的胶原—丝素蛋白真皮支架的构建及初步评价
发布时间：2018-08-01 08:49
【摘要】：目的构建功能化组织工程真皮支架——负载纳米银的胶原-丝素蛋白真皮支架(collagen-fibroin scaffolds loaded with silver nanoparticles,NAg-CFS),并对其微观结构及生物学性能进行初步评价,为纳米银在组织工程真皮中的应用寻找新的载体及可行的方式。方法通过胃蛋白酶消化与酸抽提相结合的方法,从牛跟腱中获取Ⅰ型胶原。通过脱胶、盐析、透析等步骤从桑蚕蚕茧中获取丝素蛋白。将5mg/mL的胶原蛋白和5mg/mL的丝素蛋白以1:1的体积比配制成混合溶液,随后将10mmg/mL的纳米银溶液与胶原-丝素蛋白溶液以1:1000的体积比混匀,通过冷冻-冻干法制备出负载纳米银的胶原-丝素蛋白真皮支架(NAg-CFS)和不负载纳米银的胶原-丝素蛋白真皮支架(CFS)。以NAg-CFS为实验组,CFS为对照组,用扫描电子显微镜观察上述两种支架的微观结构。取18只雄性SD大鼠,在每只大鼠背部脊柱中线两侧制作4个直径为2cm的圆形全层皮肤缺损创面,在同一只大鼠背部创面交叉移植两种支架(NAg-CFS和CFS),行同体对照,缝合后加压包扎。在术后第7、14、28天,分别取6只SD大鼠,对创面进行观察、拍照,获取组织标本后过量麻醉处死。通过苏木素/伊红(HE)染色观察创面炎性细胞浸润和真皮再生情况,通过CD68免疫组织化学染色显示创面的巨噬细胞,并用RT-PCR技术检测创面IL-6、IL-10的mRNA相对表达水平。采用Student-t检验对数据进行统计分析。结果(1)扫描电镜观察结果显示,CFS和NAg-CFS的微观结构相似。(2)大鼠创面大体观察的结果显示,实验组创面周围炎症反应较轻,且支架与组织结合更牢固;而对照组创面周围的炎症反应较强,支架更容易脱落。(3)苏木素/伊红(HE)染色结果显示,在术后观察的各个时相点,与对照组相比,均可见实验组支架及创面内部的炎症细胞较少、新生组织生长速度较快。(4)CD68免疫组织化学染色及阳性细胞计数结果显示,在术后第14和第28天,实验组支架及创面内部的巨噬细胞浸润数量显著少于对照组。(5)RT-PCR结果显示,在术后第7、14、28天,在实验组创面上,促炎因子IL-6的mRNA相对表达量均低于对照组,且均有显著性差异;而实验组创面抗炎因子IL-10的mRNA相对表达量在术后第14天明显高于对照组,具有统计学意义(p0.05),在第7天高于对照组但无显著性差异。结论本实验成功构建了负载纳米银的胶原-丝素蛋白真皮支架,并初步探讨该支架在大鼠移植后的变化和可能机制。结果表明该真皮支架植入创面后,可以缓解支架本身及其周围创面组织的炎症反应。
[Abstract]:Objective to construct a functional tissue engineering dermal scaffold (collagen-fibroin scaffolds loaded with silver nanoparticles-NAg-CFS) loaded with nano-silver, and to evaluate its microstructure and biological properties. To find a new carrier and feasible way for the application of nano-silver in tissue engineering dermis. Methods Type I collagen was obtained from bovine Achilles tendon by pepsin digestion and acid extraction. Silk fibroin was obtained from silkworm cocoon by degumming, salting out and dialysis. The collagen of 5mg/mL and the silk fibroin of 5mg/mL were mixed into a mixture solution at 1:1 volume, and then the nano-silver solution of 10mmg/mL and the collagen-fibroin solution were mixed at 1: 1000 volume. Collagen fibroin dermal scaffold (NAg-CFS) and collagen fibroin dermal scaffold (CFS).) without silver nanoparticles were prepared by freeze-drying method. The microstructures of the above two scaffolds were observed by scanning electron microscope (SEM). Eighteen male Sprague-Dawley rats were used to make 4 round full-thickness skin defect wounds with diameter of 2cm on both sides of the midline of the dorsal spine of each rat. Two kinds of scaffolds (NAg-CFS and CFS),) were cross-transplanted in the same rat back wound. Six SD rats were taken at the 14th day after operation. The wound was observed and photographed, and the tissue specimens were collected and killed under excessive anesthesia. The infiltration of inflammatory cells and dermal regeneration were observed by hematoxylin / eosin (HE) staining. The macrophages of the wound were detected by CD68 immunohistochemical staining. The relative expression of IL-6 / IL-10 was detected by RT-PCR technique. The data were analyzed by Student-t test. Results (1) the results of scanning electron microscope showed that the microstructure of NAg-CFS and CFS were similar. (2) the results of gross observation showed that the inflammatory reaction around the wound was lighter in the experimental group and the stents were more firmly bound to the tissue. In the control group, the inflammatory reaction around the wound was stronger, and the stent was more easily shed. (3) the results of hematoxylin / eosin (HE) staining showed that, compared with the control group, There were fewer inflammatory cells and faster growth rate of new tissue in the experimental group. (4) the results of CD68 immunohistochemical staining and positive cell count showed that on the 14th and 28th day after operation, the inflammatory cells in the scaffold and wound surface of the experimental group were less, and the growth rate of the new tissue was faster. The number of macrophages infiltrated in the scaffold and wound in the experimental group was significantly less than that in the control group. (5) the results of RT-PCR showed that the relative expression of mRNA of pro-inflammatory factor IL-6 on the wounds of the experimental group was lower than that of the control group on the 714 ~ (th) day after operation, and there was significant difference between the two groups. The relative expression of anti-inflammatory factor IL-10 in the experimental group was significantly higher than that in the control group on the 14th day after operation (p0.05), and on the 7th day it was higher than that in the control group, but there was no significant difference. Conclusion the collagen-fibroin dermal scaffold loaded with nano-silver was successfully constructed in this experiment, and the changes and possible mechanism of the scaffold after transplantation in rats were discussed. The results showed that the inflammatory reaction of the stent itself and its surrounding wound tissue could be alleviated after the implantation of the dermal scaffold.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R641
，
本文编号：2157036
[Abstract]:Objective to construct a functional tissue engineering dermal scaffold (collagen-fibroin scaffolds loaded with silver nanoparticles-NAg-CFS) loaded with nano-silver, and to evaluate its microstructure and biological properties. To find a new carrier and feasible way for the application of nano-silver in tissue engineering dermis. Methods Type I collagen was obtained from bovine Achilles tendon by pepsin digestion and acid extraction. Silk fibroin was obtained from silkworm cocoon by degumming, salting out and dialysis. The collagen of 5mg/mL and the silk fibroin of 5mg/mL were mixed into a mixture solution at 1:1 volume, and then the nano-silver solution of 10mmg/mL and the collagen-fibroin solution were mixed at 1: 1000 volume. Collagen fibroin dermal scaffold (NAg-CFS) and collagen fibroin dermal scaffold (CFS).) without silver nanoparticles were prepared by freeze-drying method. The microstructures of the above two scaffolds were observed by scanning electron microscope (SEM). Eighteen male Sprague-Dawley rats were used to make 4 round full-thickness skin defect wounds with diameter of 2cm on both sides of the midline of the dorsal spine of each rat. Two kinds of scaffolds (NAg-CFS and CFS),) were cross-transplanted in the same rat back wound. Six SD rats were taken at the 14th day after operation. The wound was observed and photographed, and the tissue specimens were collected and killed under excessive anesthesia. The infiltration of inflammatory cells and dermal regeneration were observed by hematoxylin / eosin (HE) staining. The macrophages of the wound were detected by CD68 immunohistochemical staining. The relative expression of IL-6 / IL-10 was detected by RT-PCR technique. The data were analyzed by Student-t test. Results (1) the results of scanning electron microscope showed that the microstructure of NAg-CFS and CFS were similar. (2) the results of gross observation showed that the inflammatory reaction around the wound was lighter in the experimental group and the stents were more firmly bound to the tissue. In the control group, the inflammatory reaction around the wound was stronger, and the stent was more easily shed. (3) the results of hematoxylin / eosin (HE) staining showed that, compared with the control group, There were fewer inflammatory cells and faster growth rate of new tissue in the experimental group. (4) the results of CD68 immunohistochemical staining and positive cell count showed that on the 14th and 28th day after operation, the inflammatory cells in the scaffold and wound surface of the experimental group were less, and the growth rate of the new tissue was faster. The number of macrophages infiltrated in the scaffold and wound in the experimental group was significantly less than that in the control group. (5) the results of RT-PCR showed that the relative expression of mRNA of pro-inflammatory factor IL-6 on the wounds of the experimental group was lower than that of the control group on the 714 ~ (th) day after operation, and there was significant difference between the two groups. The relative expression of anti-inflammatory factor IL-10 in the experimental group was significantly higher than that in the control group on the 14th day after operation (p0.05), and on the 7th day it was higher than that in the control group, but there was no significant difference. Conclusion the collagen-fibroin dermal scaffold loaded with nano-silver was successfully constructed in this experiment, and the changes and possible mechanism of the scaffold after transplantation in rats were discussed. The results showed that the inflammatory reaction of the stent itself and its surrounding wound tissue could be alleviated after the implantation of the dermal scaffold.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R641
，
本文编号：2157036
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