本文選題：糖尿病潰瘍 + 水凝膠��； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：目的隨著糖尿病及其主要并發(fā)癥糖尿病足潰瘍的發(fā)病率日益增高,為改善其所造成的經濟損失及截肢等后果,本研究旨在合成一種類似于細胞外基質的新型功能化復合水凝膠材料,通過負載白細胞介素4(IL-4)對巨噬細胞進行免疫調控,誘導其分化為選擇性激活的巨噬細胞即M2亞型,從而分泌抗炎性細胞因子發(fā)揮抗炎性作用,以促進糖尿病足潰瘍傷口的快速愈合。方法以殼聚糖和葡聚糖為主要原料合成巰基化殼聚糖(CS-SH)和馬來酸葡聚糖(Dex-Ma),再通過Michael加成反應制備得到新型功能化復合水凝膠即巰基化殼聚糖/馬來酸葡聚糖(CS-SH/Dex-Ma,CSDM)復合水凝膠。通過調節(jié)巰基含量與馬來酸含量構筑一系列不同比例的水凝膠,利用核磁共振譜儀和傅里葉紅外光譜儀分析其結構,運用掃描電子顯微鏡(SEM)觀察該凝膠的微觀結構,并對其溶脹動力學和流變學等特性進行測定評價,同時運用四甲基偶氮唑鹽(MTT)法評估水凝膠的生物相容性。基于上述關于水凝膠的基本性能評價后,進一步對水凝膠進行負載IL-4藥物實驗,同時用該載藥水凝膠對小鼠單核巨噬細胞白血病細胞(RAW264.7)進行干預評價,實驗設立五組,分別為正常對照(NC)組、脂多糖(LPS)組、IL-4組、水凝膠(CSDM)組及水凝膠+IL-4(CSDM+IL-4)組,于顯微鏡下對干預后的各組細胞形態(tài)進行觀察并拍照記錄,利用總一氧化氮檢測試劑盒測定干預后各組細胞上清液中的NO含量,酶聯免疫吸附試驗(ELISA)檢測水凝膠及藥物干預細胞后各組上清液中腫瘤壞死因子a(TNF-a)和白細胞介素10(IL-10)的分泌情況,以及通過實時定量多聚酶鏈反應(qPCR)檢測干預后各組的RAW264.7巨噬細胞中誘導型一氧化氮合酶(iNOS)和精氨酸酶-1(Arg-1)的相對表達情況。結果通過調節(jié)巰基含量與馬來酸含量制備出5種比例的水凝膠,即巰基與雙鍵的摩爾比分別為1:4、1:2、1:1、2:1和4:1(CS1DM4、CS1DM2、CS1DM1、CS2DM1、CS4DM1),通過核磁與紅外測試均可證明水凝膠成功合成,SEM下觀察水凝膠具有三維多孔狀結構,孔徑為5μm左右,該結構為水凝膠發(fā)揮其功能的基礎條件;同時這種多孔結構可使水凝膠的溶脹率在60min內基本達到溶脹平衡,溶脹率高達為400%,隨著時間的延長,水凝膠的溶脹率持續(xù)升高,最高達700%,結果顯示該凝膠具有良好的吸水功能;水凝膠的流變學結果顯示,隨著角頻率的逐漸增長(0.1~100 rad/s),所得水凝膠的G'值均大于G"值,即儲存模量大于損耗模量,說明水凝膠的結構穩(wěn)定;MTT結果表明,水凝膠浸出液分別與NIH3T3細胞和RAW264.7巨噬細胞培養(yǎng)24h和48h后,浸出液濃度4mg/mL的水凝膠浸出液所培養(yǎng)的細胞存活率均在80%以上,說明該凝膠具有良好的生物相容性,毒副作用小。對細胞進行干預后,發(fā)現NC組的細胞形態(tài)為圓形、小而透亮的正常形態(tài),經LPS誘導后表現為非常態(tài)的多邊形并有偽足,而IL-4誘導后細胞形態(tài)無明顯變化,并且CSDM組以及CSDM+IL-4組對RAW264.7巨噬細胞的形態(tài)也沒有明顯的影響;NO測試結果表明CSDM+IL-4組的NO釋放量明顯低于CSDM組;ELISA檢測TNF-a的分泌結果顯示CSDM+IL-4組較CSDM組顯著降低,CSDM+IL-4組細胞分泌IL-10明顯高于CSDM組;通過qPCR檢測結果顯示CSDM+IL-4組的iNOS相對表達量顯著低于CSDM組,而Arg-1的表達量CSDM+IL-4組較CSDM組顯著升高。結論CSDM復合水凝膠具有作為傷口敷料的基本條件和良好的生物相容性,不僅為傷口組織提供優(yōu)良的濕潤環(huán)境,同時還可以吸收多余的炎性滲液,負載IL-4后可以成功使巨噬細胞誘導分化為M2型巨噬細胞,從而使抗炎性細胞因子IL-10的分泌量顯著升高,Arg-1的mRNA表達量也明顯增高,以發(fā)揮抗炎性作用,促進糖尿病足潰瘍的快速愈合。
[Abstract]:Objective with diabetes and its major complications, the incidence of diabetic foot ulcers is increasing. In order to improve the consequences of economic loss and amputation, this study aims to synthesize a new type of functional composite hydrogel, which is similar to the extracellular matrix, and to regulate the immunoregulation of macrophages through the load of interleukin 4 (IL-4). It is induced to differentiate into a selectively activated macrophage, M2 subtype, which secretes anti-inflammatory cytokines to promote the rapid healing of diabetic foot ulcers. Methods chitosan and glucan are used as the main ingredients to synthesize mercapted chitosan (CS-SH) and maleic acid glucan (Dex-Ma), and then Michael addition reaction. A new functional composite hydrogel, mercapto chitosan / CS-SH/Dex-Ma (CSDM) hydrogels, was prepared. By adjusting the content of the sulfhydryl group and the content of maleic acid, a series of different hydrogels were constructed. The structure of the hydrogels were analyzed by NMR and FTIR, and the scanning electron microscope (SEM) was used. The microstructure of the gel was observed and the swelling kinetics and rheological properties were measured and evaluated. The biocompatibility of the hydrogel was evaluated by four methyl azazolide (MTT) method. Based on the evaluation of the basic properties of the hydrogels, the hydrogel was further loaded with IL-4 medicine and the hydrogel was used. The mice mononuclear macrophage leukemia cell (RAW264.7) was evaluated. The experiment set up five groups, which were normal control (NC) group, lipopolysaccharide (LPS) group, IL-4 group, hydrogel (CSDM) group and hydrogel +IL-4 (CSDM+IL-4) group. Under the microscope, the cell morphology of each group was observed and photographed, and the total nitric oxide was detected by the total nitric oxide. The content of NO in the cell supernatant after intervention was measured by the kit, and the enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor A (TNF-a) and interleukin 10 (IL-10) in the supernatant of each group after the hydrogel and drug intervention cells, and the RAW264.7 of each group after intervention by real-time fixed polymerase chain reaction (qPCR). The relative expression of inducible nitric oxide synthase (iNOS) and arginase -1 (Arg-1) in macrophages. Results by adjusting the sulfhydryl content and maleic acid content, 5 kinds of hydrogels were prepared, that is, the mole ratio of the sulfhydryl and double bonds is 1:4,1:2,1:1,2:1 and 4:1 (CS1DM4, CS1DM2, CS1DM1, CS2DM1, CS4DM1), by NMR and IR test. The hydrogels can be successfully synthesized. The hydrogels have a three-dimensional multi pore structure under SEM. The pore size is about 5 mu m. This structure is the basic condition for the hydrogel to play its function. At the same time, this porous structure can make the swelling rate of hydrogel basically reach the swelling balance in 60min, the swelling rate is up to 400%, with the time prolonging, hydrogel. The swelling rate continued to rise, up to 700%. The results showed that the gel had a good water absorption function. The rheological results of the hydrogel showed that the G'value of the hydrogel was greater than the value of G with the gradual increase of the angular frequency (0.1~100 rad/s), that is, the storage modulus was greater than the loss modulus, indicating that the structure of the hydrogel was stable; the MTT results showed that the hydrogel was leached. After 24h and 48h were cultured with NIH3T3 cells and RAW264.7 macrophages respectively, the survival rate of the cells cultured in the leaching solution of 4mg/mL was above 80%, indicating that the gel had good biocompatibility and small side effects. The cell morphology of the NC group was round, small and bright. State, after LPS induction, the expression of the polygon and the pseudo foot, and the cell morphology without obvious change after IL-4 induction, and the CSDM group and CSDM+IL-4 group have no obvious influence on the morphology of RAW264.7 macrophage. The NO test results showed that the NO release of CSDM+IL-4 group was obviously lower than that of the CSDM group; ELISA test TNF-a secretion showed CS. Group DM+IL-4 was significantly lower than group CSDM, and the secretion of IL-10 in group CSDM+IL-4 was significantly higher than that in group CSDM. The relative expression of iNOS in group CSDM+IL-4 was significantly lower than that in group CSDM, and the expression of Arg-1 in CSDM+IL-4 group was significantly higher than that in CSDM group. Conclusion the composite hydrogel has a basic condition and good survival as a wound dressing. The compatibility, not only provides excellent moist environment for wound tissue, but also absorbs superfluous inflammatory infiltration fluid. After loading IL-4, macrophages can be induced to differentiate into M2 type macrophages, thus the secretion of anti-inflammatory cytokine IL-10 is increased significantly, and the expression of mRNA in Arg-1 is also significantly increased in order to play an anti inflammatory effect. It promotes the rapid healing of diabetic foot ulcers.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2

【參考文獻】

相關期刊論文 前1條

1 Antonino Tuttolomondo;Carlo Maida;Antonio Pinto;;Diabetic foot syndrome:Immune-inflammatory features as possible cardiovascular markers in diabetes[J];World Journal of Orthopedics;2015年01期

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本文編號：2028048