载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响  

Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats

作　　者：张清荣 陈长友 徐娜 吕大伦 贾杰只 李文红 罗高兴 于云龙 张逸[1] Zhang Qingrong;Chen Changyou;Xu Na;Lyu Dalun;Jia Jiezhi;Li Wenhong;Luo Gaoxing;Yu Yunlong;Zhang Yi(Department of Burns and Plastic Surgery,Affiliated Hospital of Nantong University,Nantong 226001,China;State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burn Research,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing Key Laboratory for Disease Proteomics,Chongqing 400038,China;Department of Burns and Plastic Surgery,the First Affiliated Hospital of Wannan Medical College,Wuhu 241001,China;Beijing Jayyalife Biological Technology Company,Beijing 100094,China)

机构地区：[1]南通大学附属医院烧伤整形外科,南通226001 [2]陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,重庆400038 [3]皖南医学院第一附属医院烧伤整形外科,芜湖241001 [4]北京桀亚莱福生物技术有限责任公司,北京100094

出　　处：《中华烧伤与创面修复杂志》2022年第10期914-922,共9页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金国际(地区)合作与交流项目(81920108022);南通市科技项目(JC2021178,HS2020006);中国高校产学研创新基金(2021JH033)。

摘　　要：目的探讨载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响。方法采用实验研究方法。通过油包水乳化法制备聚乙烯醇/海藻酸钠微球(单纯微球)、P311微球及异硫氰酸荧光素标记的牛血清白蛋白(FITC-BSA)微球,于光学显微镜/倒置荧光显微镜下观察形貌。制备壳聚糖溶液,将壳聚糖溶液和β-磷酸甘油二钠水合物混合制备单纯温敏水凝胶,在单纯温敏水凝胶中加入相应物质制备载单纯微球和载P311微球的温敏水凝胶,观察4种液体在37℃时倾斜状态下的形态变化,冷冻干燥后于扫描电子显微镜下观察微观形貌。取18只3~4周龄雄性SD大鼠,分为不进行任何处理的正常组及于背部脊柱两侧分别制作1个全层皮肤缺损创面并进行相应处理的敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组,每组3只。取5组全层皮肤缺损大鼠,于伤后0(即刻)、5、10、15 d观察创面愈合情况,计算伤后5、10、15 d创面愈合率;取5组全层皮肤缺损大鼠伤后15 d创面和创缘组织及正常组大鼠相同部位正常皮肤组织,行苏木精-伊红染色观察组织学变化,行免疫组织化学染色观测CD31及血管内皮生长因子(VEGF)的表达,采用蛋白质印迹法检测CD31及VEGF的蛋白表达。样本数均为3。对数据行单因素方差分析、重复测量方差分析及Bonferroni校正。结果单纯微球呈球形,表面疏松多孔;P311微球及FITC-BSA微球表面光滑无孔隙,且FITC-BSA微球散发出均匀的绿色荧光;3种微球直径基本一致,为33.1~37.7μm。在37℃时倾斜状态下,与壳聚糖溶液及单纯温敏水凝胶相比,载微球的2种水凝胶结构更稳定。载微球的2种水凝胶网状结构较壳聚糖溶液、单纯温敏水凝胶更致密,且其横断面可见直径约30μm的微球。伤后15 d内,5组大鼠创面均不同程度愈合,其中载P311微球水凝胶组大鼠创面愈合情况最好。�Objective To explore the effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats.Methods The method of experimental study was adopted.The polyvinyl alcohol/sodium alginate microspheres(simple microspheres),P311 microspheres,and bovine serum albumin labeled with fluorescein isothiocyanate(FITC-BSA)microspheres were prepared by water-in-oil emulsification,and then their morphology was observed under a light microscope/inverted fluorescence microscope.Chitosan solution was prepared,chitosan solution andβ-glycerol phosphate disodium hydrate were mixed to prepare simple thermosensitive hydrogels,and thermosensitive hydrogels loaded with simple microspheres or P311 microspheres were prepared by adding corresponding substances in simple thermosensitive hydrogels.The morphological changes of the prepared four liquids in the state of tilt was observed at 37℃.After being freeze-dried,the micromorphology of the prepared four liquids was observed under a scanning electron microscope.Eighteen 3-4-week-old male Sprague-Dawley rats were divided into normal group without any treatment,dressing group,chitosan group,hydrogel alone group,simple microspheres-loaded hydrogel group,and P311 microspheres-loaded hydrogel group,which were inflicted with one full-thickness skin defect wound on both sides of the back spine and were dealt correspondingly,with 3 rats in each group.Rats with full-thickness skin defects in the five groups were collected,the wound healing was observed on post injury day(PID)0(immediately),5,10,and 15,and the wound healing rates on PID 5,10,and 15 were calculated.The wound and wound margin tissue of rats with full-thickness skin defects in the five groups on PID 15 and normal skin tissue in the same site of rats in normal group were collected,hematoxylin and eosin staining was conducted to observe the histological changes,immunohistochemical staining was performed to observe the expressions of CD31 and vascular endothelial growth factor(

关 键 词：生物相容性材料 水凝胶 微球体 P311 血管生成 创面修复 

分 类 号：R622[医药卫生—整形外科]