Pluronic F127水凝胶负载脂肪干细胞对糖尿病性溃疡血管生成及愈合作用的研究
发布时间：2019-01-28 07:37
【摘要】：研究背景与目的:糖尿病难愈性溃疡是糖尿病患者最严重、治疗费用最高的慢性并发症之一。相较于正常创面,糖尿病性溃疡往往迁延不愈,其最具特征性的变化是血管发生受损和肉芽组织形成障碍。目前临床及动物实验均显示干细胞疗法可以促进创面愈合,然而,干细胞移植到糖尿病难愈性创面面临着易被原位清除、移植效率低等难题。如何将干细胞递送到糖尿病伤口部位同时保持干细胞的高存活率是干细胞移植治疗糖尿病性溃疡的一个关键挑战。研究表明,采用新型智能水凝胶作为三维支架理化模拟细胞外基质,为干细胞移植到宿主创面提供合适的载体是这一问题一种有效的解决途径。Pluronic F127为聚氧乙烯聚丙乙烯三嵌段聚合物,其水凝胶溶液具有独特的温敏特性,在一定浓度和温度下可由液态转变为多孔三维结构的凝胶态。因其具有医用可注射性及良好的生物相容性,已被美国FDA批准用于人体。本实验以糖尿病大鼠为实验对象,选取Pluronic F127水凝胶负载脂肪源性干细胞创新性应于糖尿病创面复杂的修复环境,观察其对大鼠皮肤创面新生血管形成和愈合作用的影响。方法:第1部分:胶原酶消化法分离培养健康SD大鼠ADSCs并流式鉴定细胞表型。建立SD大鼠1型糖尿病模型,MTT法比较糖尿病大鼠和正常大鼠ADSCs的增殖能力以选择合适的干细胞用于后续实验。第2部分,制备Pluronic F127水凝胶并测定凝胶化时间,CCK-8法检测不同浓度PluronicF127对ADSCs增殖的影响,选择细胞亲和性最佳的凝胶浓度包裹ADSCs制备Pluronic F127-ADSCs复合物。在成模的24只糖尿病大鼠背部对称制作2个直径9mm的全层皮肤切口并随机分为ADSCs/Gel组、ADSCs组、Gel组、PBS对照组4组(n=6)。分别注射100ul相应溶液于创面。术后观察创面大体情况并评估各组创面愈合率,HE染色观察肉芽组织形成情况,CD31免疫组化染色观察新生血管情况,Ki67检测组织细胞增殖,qPCR检测VEGF、TGFβ-1的表达。结果:成功分离纯化并流式鉴定ADSCs,MTT结果显示正常ADSCs较糖尿病来源ADSCs增殖能力更好。CCK-8结果显示20%的Pluronic F127相较于其他组更适宜细胞生长。动物伤口大体观和HE染色显示ADSCs/Gel组愈合较快且组织学评分较佳,在术后14天时愈合率为(97.8±1.8)%,明显高于其他组(P=0.000)。免疫组化结果示术后7天ADSCs/Gel、ADSCs组的血管数直观较Gel、对照组丰富,对术后14天进行微血管计数ADSCs/Gel组微脉管数目为(21.0±2.4)个,明显高于 ADSCs 组的(15.7±1.4)个(P=0.000)。qPCR 结果也显示实验组VEGF、TGFβ-1水平在术后7、14天时出现了相应上调(P0.05)。免疫荧光结果示ADSCs/Gel组在术后7天时Ki67阳性表达率多于其他三组。结论:局部移植同种异体ADSCs通过PluronicF127水凝胶的包裹能提高细胞移植到糖尿病性溃疡创面的效率,从而优化了 ADSCs促进糖尿病性溃疡血管生成和创面愈合的作用。
[Abstract]:Background & objective: diabetic refractory ulcer is one of the most serious and costly chronic complications in diabetic patients. Compared with normal wounds, diabetic ulcers are often prolonged, the most characteristic changes are vascular damage and granulation tissue formation disorder. At present, both clinical and animal experiments have shown that stem cell therapy can promote wound healing. However, stem cell transplantation to the refractory wound of diabetes is faced with problems such as easy to be removed in situ and low transplantation efficiency. How to deliver stem cells to diabetic wounds while maintaining the high survival rate of stem cells is a key challenge for stem cell transplantation in the treatment of diabetic ulcer. The results showed that the novel intelligent hydrogel was used as the physical and chemical simulation extracellular matrix of three-dimensional scaffold. It is an effective way to solve this problem by providing suitable carrier for transplantation of stem cells to host wound. Pluronic F127 is a polyoxyethylene polypropylene-triblock polymer, and its hydrogel solution has unique temperature-sensitive properties. The gel state of porous three-dimensional structure can be changed from liquid state to porous three-dimensional structure at a certain concentration and temperature. Because of its injectable medical and good biocompatibility, it has been approved for human body by the United States FDA. In this study, diabetic rats were selected to study the effects of Pluronic F127 hydrogel loaded with adipose derived stem cells on the formation and healing of neovascularization in diabetic wounds. Methods: part 1: ADSCs of healthy SD rats was isolated by collagenase digestion and identified by flow cytometry. The model of type 1 diabetes in SD rats was established. The proliferative ability of ADSCs in diabetic rats and normal rats was compared by MTT method in order to select suitable stem cells for subsequent experiments. In the second part, Pluronic F127 hydrogel was prepared and the gelation time was determined. The effect of different concentration of PluronicF127 on the proliferation of ADSCs was detected by CCK-8 method. ADSCs with the best cell affinity was selected to prepare Pluronic F127-ADSCs complex. Two full-thickness skin incisions with diameter 9mm were made on the back of 24 diabetic rats and randomly divided into ADSCs/Gel group, ADSCs group, Gel group and PBS control group (n = 4). The corresponding solution of 100ul was injected into the wound. The wound healing rate, granulation tissue formation, neovascularization, Ki67 and VEGF,TGF 尾 -1 expression were observed by HE staining, CD31 immunohistochemical staining, Ki67 and qPCR respectively. Results: ADSCs,MTT showed that normal ADSCs had better proliferative ability than ADSCs derived from diabetes, and CCK-8 showed that 20% of Pluronic F127 was more suitable for cell growth than other groups. Gross observation and HE staining showed that the healing rate of the ADSCs/Gel group was (97.8 卤1.8)% on the 14th day after operation, which was significantly higher than that of the other groups (P0. 000). Immunohistochemical results showed that the number of blood vessels in the ADSCs/Gel,ADSCs group was significantly higher than that in the Gel, control group on the 7th day after operation, and the number of microvessels in the ADSCs/Gel group was (21.0 卤2.4) on the 14th day after operation. The level of VEGF,TGF 尾 -1 in experimental group was significantly higher than that in ADSCs group (15.7 卤1.4) (P < 0.05). The results of 0.000). QPCR also showed that the level of VEGF,TGF 尾 -1 in the experimental group was up-regulated on the 7th and 14th day after operation (P0.05). Immunofluorescence results showed that the positive expression rate of Ki67 in ADSCs/Gel group was higher than that in other three groups 7 days after operation. Conclusion: local transplantation of allogeneic ADSCs can improve the efficiency of cell transplantation to diabetic ulcer wound by encapsulating PluronicF127 hydrogel, thus optimizing the role of ADSCs in promoting angiogenesis and wound healing of diabetic ulcer.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2
本文编号：2416843
[Abstract]:Background & objective: diabetic refractory ulcer is one of the most serious and costly chronic complications in diabetic patients. Compared with normal wounds, diabetic ulcers are often prolonged, the most characteristic changes are vascular damage and granulation tissue formation disorder. At present, both clinical and animal experiments have shown that stem cell therapy can promote wound healing. However, stem cell transplantation to the refractory wound of diabetes is faced with problems such as easy to be removed in situ and low transplantation efficiency. How to deliver stem cells to diabetic wounds while maintaining the high survival rate of stem cells is a key challenge for stem cell transplantation in the treatment of diabetic ulcer. The results showed that the novel intelligent hydrogel was used as the physical and chemical simulation extracellular matrix of three-dimensional scaffold. It is an effective way to solve this problem by providing suitable carrier for transplantation of stem cells to host wound. Pluronic F127 is a polyoxyethylene polypropylene-triblock polymer, and its hydrogel solution has unique temperature-sensitive properties. The gel state of porous three-dimensional structure can be changed from liquid state to porous three-dimensional structure at a certain concentration and temperature. Because of its injectable medical and good biocompatibility, it has been approved for human body by the United States FDA. In this study, diabetic rats were selected to study the effects of Pluronic F127 hydrogel loaded with adipose derived stem cells on the formation and healing of neovascularization in diabetic wounds. Methods: part 1: ADSCs of healthy SD rats was isolated by collagenase digestion and identified by flow cytometry. The model of type 1 diabetes in SD rats was established. The proliferative ability of ADSCs in diabetic rats and normal rats was compared by MTT method in order to select suitable stem cells for subsequent experiments. In the second part, Pluronic F127 hydrogel was prepared and the gelation time was determined. The effect of different concentration of PluronicF127 on the proliferation of ADSCs was detected by CCK-8 method. ADSCs with the best cell affinity was selected to prepare Pluronic F127-ADSCs complex. Two full-thickness skin incisions with diameter 9mm were made on the back of 24 diabetic rats and randomly divided into ADSCs/Gel group, ADSCs group, Gel group and PBS control group (n = 4). The corresponding solution of 100ul was injected into the wound. The wound healing rate, granulation tissue formation, neovascularization, Ki67 and VEGF,TGF 尾 -1 expression were observed by HE staining, CD31 immunohistochemical staining, Ki67 and qPCR respectively. Results: ADSCs,MTT showed that normal ADSCs had better proliferative ability than ADSCs derived from diabetes, and CCK-8 showed that 20% of Pluronic F127 was more suitable for cell growth than other groups. Gross observation and HE staining showed that the healing rate of the ADSCs/Gel group was (97.8 卤1.8)% on the 14th day after operation, which was significantly higher than that of the other groups (P0. 000). Immunohistochemical results showed that the number of blood vessels in the ADSCs/Gel,ADSCs group was significantly higher than that in the Gel, control group on the 7th day after operation, and the number of microvessels in the ADSCs/Gel group was (21.0 卤2.4) on the 14th day after operation. The level of VEGF,TGF 尾 -1 in experimental group was significantly higher than that in ADSCs group (15.7 卤1.4) (P < 0.05). The results of 0.000). QPCR also showed that the level of VEGF,TGF 尾 -1 in the experimental group was up-regulated on the 7th and 14th day after operation (P0.05). Immunofluorescence results showed that the positive expression rate of Ki67 in ADSCs/Gel group was higher than that in other three groups 7 days after operation. Conclusion: local transplantation of allogeneic ADSCs can improve the efficiency of cell transplantation to diabetic ulcer wound by encapsulating PluronicF127 hydrogel, thus optimizing the role of ADSCs in promoting angiogenesis and wound healing of diabetic ulcer.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2
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相关期刊论文 前3条
1 Adriana Georgescu;;Vascular dysfunction in diabetes: The endothelial progenitor cells as new therapeutic strategy[J];World Journal of Diabetes;2011年06期
2 Louis Casteilla;Valérie Planat-Benard;Patrick Laharrague;Béatrice Cousin;;Adipose-derived stromal cells: Their identity and uses in clinical trials, an update[J];World Journal of Stem Cells;2011年04期
3 陆颖理,胡申江,沈周俊,邵一川;Changes of macrovascular endothelial ultrastructure and gene expression of endothelial nitric oxide synthase in diabetic rats[J];Chinese Medical Journal;2004年08期
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