摘要
目的探讨负载大鼠表皮干细胞(ESC)的聚己内酯-乙酸纤维素(PCL-CA)纳米纤维支架对大鼠全层皮肤缺损创面愈合的影响及其机制。方法采用实验研究方法。采用快速贴壁法从30只1-3 d龄SD大鼠(雌雄不明)中分离培养原代ESC,采用流式细胞仪、免疫荧光法分别鉴定原代细胞中整合素β_(1)、细胞角蛋白19(CK19)为阳性表达后,采用第1代ESC进行后续实验。采用静电纺丝技术制备以聚己内酯和乙酸纤维素为组分的PCL-CA纳米纤维支架,采用扫描电子显微镜观察支架拓扑结构并测量其中25条纤维直径。采用构建的PCL-CA纳米纤维支架作为培养基底,使用角质形成细胞(KC)培养基培养ESC构建ESC-纳米纤维支架复合物(下称ESC支架),培养3 d,采用扫描电子显微镜观察支架中ESC的形态及其与支架的关系。将ESC支架中的ESC作为PCL-CA纳米纤维支架组,将在Ⅳ型胶原包被的培养皿中使用KC培养基培养的ESC作为Ⅳ型胶原组,培养3 d,采用蛋白质印迹法检测2组ESC中CK19的蛋白表达水平(样本数为3);培养7 d,采用免疫荧光法检测2组ESC中CK19和增殖细胞核抗原(PCNA)的蛋白表达。在15只6-8周龄雄性SD大鼠背部左右两侧均制备1个直径约2 cm的圆形全层皮肤缺损创面后,将大鼠按随机数字表法等分为不行植入的空白对照组、植入PCL-CA纳米纤维支架的单纯支架组、植入培养3 d构建的ESC支架的ESC支架组,计算伤后3、7、14、21 d创面面积百分率(样本数为5);取伤后21 d创缘新生皮肤组织,行Masson染色后评估创面愈合质量,采用蛋白质印迹法检测Notch信号通路关键蛋白Notch1、Jagged1、Hes1的蛋白表达水平(样本数为3)。对数据行单因素方差分析、重复测量方差分析、独立样本t检验、Bonferroni校正。结果构建的PCL-CA纳米纤维支架具有疏松多孔的网格状多层立体结构,其中的纤维表面光滑无孔隙,纤维直径为(383±24)nm。培养3 d,ESC支架中�
Objective To explore the effects and mechanisms of polycaprolactone-cellulose acetate(PCL-CA)nanofiber scaffold loaded with rat epidermal stem cells(ESCs)on wound healing of full-thickness skin defects in rats.Methods The experiment research method was applied.The primary ESCs were isolated from 1-3 d old Sprague-Dawley(SD)rats(undefined gender)by rapid adherent method and cultured by rapid adherent method.ESCs of the first passage were used for the subsequent experiments after the positive expressions of integrin β_(1) and cytokeratin 19(CK19)in primary cells were identified respectively by flow cytometey and immunofluorescence method.PCL-CA nanofiber scaffolds with polycaprolactone and cellulose acetate as components were prepared by electrospinning technique.The topological structure of the nanofiber scaffolds was determined and the diameter of 25 fibers was measured by scanning electron microscope.The constructed PCL-CA nanofiber scaffolds were used as the culture substrate for ESCs,which were cultured in keratinocytes(KCs)medium to construct ESCs-nanofiber scaffold complex(hereinafter referred to as ESCs scaffold).After 3 days of culture,the morphology of ESCs in the scaffold and their relationship was observed by scanning electron microscopy.The ESCs in ESCs scaffold were set as PCL-CA nanofiber scaffold group,and the ESCs cultured with KCs medium in culture dishes coated with typeⅣcollagen were set as typeⅣcollagen group.Western blotting was used to detect the protein expression level of CK19 in ESCs in the two groups after 3 days of culture(n=3).The protein expressions of CK19 and proliferating nuclear antigen(PCNA)in ESCs in the two groups were detected by immunofluorescence method after 7 days of culture.A circular full-thickness skin wound of about 2 cm in diameter was prepared on both left and right sides of the back of 15 male SD rats aged 6-8 weeks.The rats were then equally divided into blank control group without implantation,scaffold alone group implanted with PCL-CA nanofiber scaffold,and E
作者
林志骁
张玉恒
黄容
李学拥
Lin Zhixiao;Zhang Yuheng;Huang Rong;Li Xueyong(Department of Burns and Plastic Surgery,the Second Affiliated Hospital of Air Force Medical University,Xi'an 710038,China)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2021年第5期460-468,共9页
Chinese Journal of Burns
基金
国家自然科学基金面上项目(81671929)。
