负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶的制备及其对大鼠全层皮肤缺损创面愈合的作用  

Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats

作　　者：刘颖 程凤 王泽薇 金红旭 曹滨验 游平飞 胡安 史秀云 杜娟[2] 苑志新 Liu Ying;Cheng Feng;Wang Zewei;Jin Hongxu;Cao Binyan;You Pingfei;Hu An;Shi Xiuyun;Du Juan;Yuan Zhixin(Department of Emergency Medicine,Northern Theater Command General Hospital,Shenyang 110016,China;Laboratory of Acute and Critical Care Research and Transformation,Jilin Provincial People's Hospital,Changchun 130021,China;Department of Emergency Medicine,Jilin Provincial People's Hospital,Changchun 130021,China)

机构地区：[1]北部战区总医院急诊医学科,沈阳110016 [2]吉林省人民医院急危重症科研与转化实验室,长春130021 [3]吉林省人民医院急诊医学科,长春130021

出　　处：《中华烧伤与创面修复杂志》2024年第1期50-56,共7页Chinese Journal of Burns And Wounds

基　　金：辽宁省博士科研启动基金(2023-BS-032)。

摘　　要：目的制备负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶并探讨其对大鼠全层皮肤缺损创面愈合的作用。方法该研究为实验研究。通过酸水解碱的方法提取甲壳素纳米纤维,将提取物与透明质酸和胶原混合制备甲壳素/透明质酸/胶原水凝胶(以下简称水凝胶),另制备负载小鼠脂肪干细胞的水凝胶。将30只雄性12周龄豚鼠按照随机数字表法分为阴性对照组、阳性对照组和水凝胶组(每组10只),分别在豚鼠背部两侧涂抹乙醇、4-氨基苯甲酸乙酯、前述制备的不含细胞的水凝胶进行诱导接触和激发接触,于激发接触后24、48 h观察皮肤水肿和红斑形成情况。将小鼠脂肪干细胞分为行常规培养的正常对照组和用前述制备的不含细胞的水凝胶培养的水凝胶组,培养3 d,采用蛋白质印迹法检测血小板衍生生长因子-D(PDGF-D)、胰岛素样生长因子Ⅰ(IGF-Ⅰ)、转化生长因子β1(TGF-β1)的蛋白表达(样本数均为3)。取8只雄性8周龄SD大鼠,在其背部两侧各制备1个圆形全层皮肤缺损创面,分别作为空白对照组(不进行任何处理)和水凝胶组(涂抹前述制备的负载脂肪干细胞的水凝胶)。伤后0(即刻)、2、4、8、10 d,观察创面愈合情况并计算伤后2、4、8、10 d的创面愈合率。取伤后10 d创面组织,行苏木精-伊红染色观察新生组织形成情况,采用酶联免疫吸附测定法检测白细胞介素1α(IL-1α)、IL-6、IL-4和IL-10的浓度,行免疫组织化学染色观察CD16、CD206阳性细胞表达并计算阳性细胞百分比。动物实验样本数均为8。结果激发接触后24 h,3组豚鼠皮肤均无水肿形成,仅阳性对照组7只豚鼠皮肤出现轻微红斑。激发接触后48 h,阳性对照组8只豚鼠皮肤出现红斑,4只豚鼠皮肤出现水肿;其余2组豚鼠皮肤无明显红斑或水肿。培养3 d,水凝胶组脂肪干细胞中PDGF-D、IGF-Ⅰ和TGF-β1的蛋白表达水平均明显高于正常对照组(t�Objective To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats.Methods The research was an experimental research.Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method,and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels(hereinafter referred to as hydrogels).Besides,the hydrogels loaded with mouse adipose-derived stem cells were prepared.Thirty male 12-week-old guinea pigs were divided into negative control group,positive control group,and hydrogel group according to the random number table,with 10 guinea pigs in each group.Ethanol,4-aminobenzoic acid ethyl ester,or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact,and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact.Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells.After 3 d of culture,protein expressions of platelet-derived growth factor-D(PDGF-D),insulin-like growth factor-Ⅰ(IGF-Ⅰ),and transforming growth factorβ1(TGF-β1)were detected by Western blotting(n=3).Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back.The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied.Wound healing was observed at 0(immediately),2,4,8,and 10 d after injury,and the wound healing rate was calculated at 2,4,8,and 10 d after injury.Wound tissue samples at 10 d after injury were collected,the new tissue formation was observed by hematoxylin-eosin staining;the concentrations of interleukin

关 键 词：生物相容性材料 水凝胶 炎症 巨噬细胞 脂肪干细胞 创面修复 

分 类 号：R318.08[医药卫生—生物医学工程]