濃度梯度堿性成纖維細胞生長因子對豬糖尿病潰瘍創(chuàng)面愈合的促進作用
發(fā)布時間：2018-08-01 12:31
【摘要】：背景慢性潰瘍?yōu)樘悄虿〕Ｒ姴l(fā)癥之一,糖尿病創(chuàng)面愈合延遲甚至是經(jīng)久不愈,明顯影響患者生活質(zhì)量。隨著糖尿病患病人口數(shù)量不斷增長,糖尿病相關(guān)的潰瘍以及難愈性創(chuàng)面逐漸引起世界關(guān)注。研究表示,糖尿病引起的周圍神經(jīng)病變、創(chuàng)面局部血供不良、內(nèi)源性生長因子水平降低等微環(huán)境紊亂及感染風險的增高都是延遲創(chuàng)面愈合的主要因素。許多實驗證明堿性成纖維細胞生長因子作為重要的促有絲分裂因子,可誘導多種細胞的增殖與分化,藉由加強神經(jīng)再生、血管形成及肉芽生長能治療創(chuàng)面周圍神經(jīng)病變及改善細胞生長微環(huán)境,從而達到降低感染風險,同時促進了糖尿病創(chuàng)面的愈合。堿性成纖維細胞生長因子在促進創(chuàng)面肉芽形成、上皮爬行及改善局部微循環(huán)的過程中扮演了不可或缺的角色。在一定范圍堿性成纖維細胞生長因子濃度內(nèi),細胞增殖及移行程度與濃度成正比,細胞生長有濃度靶向傾向。因此推測濃度梯度堿性成纖維細胞生長因子能較高效促進糖尿病創(chuàng)面的愈合。目的利用環(huán)形濃度梯度堿性成纖維細胞生長因子結(jié)合膠原纖維,制成具有快速靶向修復活性的支架,并應用于豬糖尿病創(chuàng)面,探討成纖維細胞、上皮細胞、血管內(nèi)皮細胞等能否在生長因子濃度梯度誘導下快速而有序地向創(chuàng)面中心增殖移行,從而完成肉芽屏障建立、上皮覆蓋,加速創(chuàng)面愈合。為提高糖尿病創(chuàng)面愈合速度和質(zhì)量提供新的思路。方法實驗分為空白組、支架組、均一濃度(低、中、高)組及濃度梯度組。濃度梯度組的制備首先以細胞細胞遷移實驗結(jié)果選定合適的堿性成纖維細胞生長因子濃度梯度范圍,將支架以同心圓環(huán)狀切割成內(nèi)、中、外三部分,對應浸入高、中、低堿性成纖維細胞生長因子溶液中,并對纖維支架性質(zhì)如吸收率、結(jié)合率及釋放率進行鑒定,之后以電子顯微鏡與CCK-8觀察各組對細胞的影響。動物實驗中,將六頭雌性迷你豬依據(jù)重量注射鏈脲佐菌素溶液,待血糖穩(wěn)定維維持在13-27 mmol/L持續(xù)一周即判斷糖尿病造模完成。每只糖尿病豬做36個圓形創(chuàng)面,隨機分成六組,分別于第7,14,21日采取標本,予迷你豬安樂死后獲取第21日標本,獲得標本以電子顯微鏡及免疫組化染色觀察創(chuàng)面新生肉芽厚度、創(chuàng)面血管生成數(shù)量及堿性成纖維細胞生長因子抗體染色。結(jié)果所有雌豬糖尿病模型造模成功,術(shù)后總共三個創(chuàng)面發(fā)生感染。術(shù)后21日可見創(chuàng)面新生肉芽厚度、創(chuàng)面血管生成數(shù)量及堿性成纖維細胞生長因子抗體染色結(jié)果,相較于空白組、對照組及均一中低濃度組,濃度梯度組有顯著差異,然而相較于均一高濃度組無顯著差異。結(jié)論僅有空白纖維支架或是帶有堿性成纖維細胞生長因子組,皆能促進糖尿病創(chuàng)面的愈合,在21天的標本結(jié)果顯示相較于空白組、對照組及均一中低濃度組,濃度梯度組有顯著差異,然而相較于均一高濃度組無顯著差異。表示濃度梯度堿性成纖維細胞生長因子對細胞移行具有靶向性,能誘導創(chuàng)面有序生長,進而達到促進糖尿病創(chuàng)面愈合的作用。其促進愈合能力接近均一高濃度組,表示濃度梯度堿性成纖維細胞生長因子能更高效促進糖尿病創(chuàng)面愈合,能以最少量的生長因子達到最佳化效果。
[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.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R632.1
，
本文編號：2157589
[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.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R632.1
，
本文編號：2157589
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