【摘要】：研究背景與目的:糖尿病難愈性潰瘍是糖尿病患者最嚴重、治療費用最高的慢性并發(fā)癥之一。相較于正常創(chuàng)面,糖尿病性潰瘍往往遷延不愈,其最具特征性的變化是血管發(fā)生受損和肉芽組織形成障礙。目前臨床及動物實驗均顯示干細胞療法可以促進創(chuàng)面愈合,然而,干細胞移植到糖尿病難愈性創(chuàng)面面臨著易被原位清除、移植效率低等難題。如何將干細胞遞送到糖尿病傷口部位同時保持干細胞的高存活率是干細胞移植治療糖尿病性潰瘍的一個關鍵挑戰(zhàn)。研究表明,采用新型智能水凝膠作為三維支架理化模擬細胞外基質,為干細胞移植到宿主創(chuàng)面提供合適的載體是這一問題一種有效的解決途徑。Pluronic F127為聚氧乙烯聚丙乙烯三嵌段聚合物,其水凝膠溶液具有獨特的溫敏特性,在一定濃度和溫度下可由液態(tài)轉變?yōu)槎嗫兹S結構的凝膠態(tài)。因其具有醫(yī)用可注射性及良好的生物相容性,已被美國FDA批準用于人體。本實驗以糖尿病大鼠為實驗對象,選取Pluronic F127水凝膠負載脂肪源性干細胞創(chuàng)新性應于糖尿病創(chuàng)面復雜的修復環(huán)境,觀察其對大鼠皮膚創(chuàng)面新生血管形成和愈合作用的影響。方法:第1部分:膠原酶消化法分離培養(yǎng)健康SD大鼠ADSCs并流式鑒定細胞表型。建立SD大鼠1型糖尿病模型,MTT法比較糖尿病大鼠和正常大鼠ADSCs的增殖能力以選擇合適的干細胞用于后續(xù)實驗。第2部分,制備Pluronic F127水凝膠并測定凝膠化時間,CCK-8法檢測不同濃度PluronicF127對ADSCs增殖的影響,選擇細胞親和性最佳的凝膠濃度包裹ADSCs制備Pluronic F127-ADSCs復合物。在成模的24只糖尿病大鼠背部對稱制作2個直徑9mm的全層皮膚切口并隨機分為ADSCs/Gel組、ADSCs組、Gel組、PBS對照組4組(n=6)。分別注射100ul相應溶液于創(chuàng)面。術后觀察創(chuàng)面大體情況并評估各組創(chuàng)面愈合率,HE染色觀察肉芽組織形成情況,CD31免疫組化染色觀察新生血管情況,Ki67檢測組織細胞增殖,qPCR檢測VEGF、TGFβ-1的表達。結果:成功分離純化并流式鑒定ADSCs,MTT結果顯示正常ADSCs較糖尿病來源ADSCs增殖能力更好。CCK-8結果顯示20%的Pluronic F127相較于其他組更適宜細胞生長。動物傷口大體觀和HE染色顯示ADSCs/Gel組愈合較快且組織學評分較佳,在術后14天時愈合率為(97.8±1.8)%,明顯高于其他組(P=0.000)。免疫組化結果示術后7天ADSCs/Gel、ADSCs組的血管數(shù)直觀較Gel、對照組豐富,對術后14天進行微血管計數(shù)ADSCs/Gel組微脈管數(shù)目為(21.0±2.4)個,明顯高于 ADSCs 組的(15.7±1.4)個(P=0.000)。qPCR 結果也顯示實驗組VEGF、TGFβ-1水平在術后7、14天時出現(xiàn)了相應上調(P0.05)。免疫熒光結果示ADSCs/Gel組在術后7天時Ki67陽性表達率多于其他三組。結論:局部移植同種異體ADSCs通過PluronicF127水凝膠的包裹能提高細胞移植到糖尿病性潰瘍創(chuàng)面的效率,從而優(yōu)化了 ADSCs促進糖尿病性潰瘍血管生成和創(chuàng)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 鹵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 鹵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 鹵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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2

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本文編號：2416843