浓度梯度碱性成纤维细胞生长因子对猪糖尿病溃疡创面愈合的促进作用

发布时间：2018-08-01 12:31

【摘要】：背景慢性溃疡为糖尿病常见并发症之一,糖尿病创面愈合延迟甚至是经久不愈,明显影响患者生活质量。随着糖尿病患病人口数量不断增长,糖尿病相关的溃疡以及难愈性创面逐渐引起世界关注。研究表示,糖尿病引起的周围神经病变、创面局部血供不良、内源性生长因子水平降低等微环境紊乱及感染风险的增高都是延迟创面愈合的主要因素。许多实验证明碱性成纤维细胞生长因子作为重要的促有丝分裂因子,可诱导多种细胞的增殖与分化,藉由加强神经再生、血管形成及肉芽生长能治疗创面周围神经病变及改善细胞生长微环境,从而达到降低感染风险,同时促进了糖尿病创面的愈合。碱性成纤维细胞生长因子在促进创面肉芽形成、上皮爬行及改善局部微循环的过程中扮演了不可或缺的角色。在一定范围碱性成纤维细胞生长因子浓度内,细胞增殖及移行程度与浓度成正比,细胞生长有浓度靶向倾向。因此推测浓度梯度碱性成纤维细胞生长因子能较高效促进糖尿病创面的愈合。目的利用环形浓度梯度碱性成纤维细胞生长因子结合胶原纤维,制成具有快速靶向修复活性的支架,并应用于猪糖尿病创面,探讨成纤维细胞、上皮细胞、血管内皮细胞等能否在生长因子浓度梯度诱导下快速而有序地向创面中心增殖移行,从而完成肉芽屏障建立、上皮覆盖,加速创面愈合。为提高糖尿病创面愈合速度和质量提供新的思路。方法实验分为空白组、支架组、均一浓度(低、中、高)组及浓度梯度组。浓度梯度组的制备首先以细胞细胞迁移实验结果选定合适的碱性成纤维细胞生长因子浓度梯度范围,将支架以同心圆环状切割成内、中、外三部分,对应浸入高、中、低碱性成纤维细胞生长因子溶液中,并对纤维支架性质如吸收率、结合率及释放率进行鉴定,之后以电子显微镜与CCK-8观察各组对细胞的影响。动物实验中,将六头雌性迷你猪依据重量注射链脲佐菌素溶液,待血糖稳定维维持在13-27 mmol/L持续一周即判断糖尿病造模完成。每只糖尿病猪做36个圆形创面,随机分成六组,分别于第7,14,21日采取标本,予迷你猪安乐死后获取第21日标本,获得标本以电子显微镜及免疫组化染色观察创面新生肉芽厚度、创面血管生成数量及碱性成纤维细胞生长因子抗体染色。结果所有雌猪糖尿病模型造模成功,术后总共三个创面发生感染。术后21日可见创面新生肉芽厚度、创面血管生成数量及碱性成纤维细胞生长因子抗体染色结果,相较于空白组、对照组及均一中低浓度组,浓度梯度组有显著差异,然而相较于均一高浓度组无显著差异。结论仅有空白纤维支架或是带有碱性成纤维细胞生长因子组,皆能促进糖尿病创面的愈合,在21天的标本结果显示相较于空白组、对照组及均一中低浓度组,浓度梯度组有显著差异,然而相较于均一高浓度组无显著差异。表示浓度梯度碱性成纤维细胞生长因子对细胞移行具有靶向性,能诱导创面有序生长,进而达到促进糖尿病创面愈合的作用。其促进愈合能力接近均一高浓度组,表示浓度梯度碱性成纤维细胞生长因子能更高效促进糖尿病创面愈合,能以最少量的生长因子达到最佳化效果。
[Abstract]:Chronic ulcer in the background is one of the common complications of diabetes. The delayed healing of diabetic wound is even unhealed, which obviously affects the quality of life of the patients. With the increasing number of people with diabetes, diabetes related ulcers and refractory wounds have gradually aroused worldwide attention. A number of experiments have proved that basic fibroblast growth factor is an important mitogenic factor, which can induce proliferation and differentiation of various cells and enhance nerve regeneration and blood vessels. The formation and growth of granulation can treat the neuropathy around the wound and improve the microenvironment of cell growth, thus reducing the risk of infection and promoting the healing of diabetic wounds. Basic fibroblast growth factor plays an indispensable role in promoting the formation of wound granulation, the creeping of epithelium and improving the local microcirculation. In a certain range of basic fibroblast growth factor concentration, the proliferation and migration of cells are proportional to the concentration, and the cell growth has a concentration target tendency. Therefore, it is suggested that the concentration gradient basic fibroblast growth factor can effectively promote the healing of diabetic wound. Combined with collagen fibers, a scaffold with fast target repair activity was made and applied to the wound of swine diabetes. It was discussed whether fibroblasts, epithelial cells, vascular endothelial cells, etc. could quickly and orderly move to the center of the wound under the induction of growth factor concentration gradient, and complete the establishment of the granulation barrier, cover the epithelium and accelerate the wound healing. Method experiment was divided into blank group, stent group, homogeneous concentration (low, medium, high) group and concentration gradient group. The preparation of concentration gradient group first selected the suitable basic fibroblast growth factor concentration gradient range by cell migration test results, and the scaffold was the same. The circle of heart was cut into inner, middle and outer three parts, corresponding to the solution of high, medium and low basic fibroblast growth factor, and the properties of the fiber scaffold, such as absorptivity, binding rate and release rate, were identified. Then the effects of each group on the cells were observed by electron microscope and CCK-8. In animal experiments, six female mini pigs were injected according to weight. The streptozotocin solution was maintained at 13-27 mmol/L for one week. Each diabetic pig was divided into six groups of 36 round wounds. The specimens were taken on the day 7,14,21, and after the mini pig was euthanized, the specimens were obtained for twenty-first days, and the specimens were obtained by electron microscopy and immunohistochemical staining. The thickness of fresh-born granulation, the quantity of angiogenesis and the staining of basic fibroblast growth factor antibody were observed. Results all the female diabetic models were successfully built, and three wounds were infected after operation. The thickness of new wound granulation, the amount of angiogenesis in the wound and the staining of basic fibroblast growth factor antibody were observed on the 21 day after operation. The results showed that there were significant differences in the concentration gradient group compared to the blank group, the control group and the mean and low concentration group, but there was no significant difference compared with the homogeneous high concentration group. Conclusion only the blank fiber scaffold or the basic fibroblast growth factor group could promote the healing of diabetic wound surface, and the results of the 21 day specimen showed that it was more than empty. In the white group, the control group and the mean and low concentration group, there was significant difference in the concentration gradient group. However, there was no significant difference in the concentration gradient group, but the concentration gradient basic fibroblast growth factor was targeted to the cell migration, which could induce the wound healing in order to promote the healing of diabetic wounds. The force is close to the homogeneous high concentration group, which indicates that the concentration gradient basic fibroblast growth factor can promote the healing of diabetic wound more efficiently, and can achieve the best effect with the smallest growth factor.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R632.1
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本文编号：2157589