摘要
目的探讨肝细胞生长因子(HGF)修饰人脂肪间充质干细胞(ADSC)对糖尿病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。取2019年12月于空军军医大学第一附属医院整形科行腹部抽脂术的1名35岁健康女性术后弃用腹部脂肪组织,采用胶原酶消化法获取呈长梭形的原代ADSC,其第3代细胞经流式细胞仪鉴定阳性表达ADSC表面标志物CD29、CD90,阴性表达CD34、CD45。取第3代ADSC进行后续实验。采用慢病毒介导的HGF转染ADSC 4 h(获得HGF修饰ADSC)后,常规培养24 h,倒置相差显微镜下观察细胞形态并计算转染率。取81只4周龄雄性SD大鼠,通过高糖高脂饮食联合链脲佐菌素注射诱导为糖尿病大鼠模型后,在每只大鼠背部制作1.5 cm×1.5 cm全层皮肤缺损创面。采用随机数字表法,将致伤大鼠分为磷酸盐缓冲液(PBS)组、单纯ADSC组、HGF修饰ADSC组,每组27只,均分别于伤后1、3、7 d在创周注射相同体积的相应物质。采用随机数字表法选取各组9只大鼠,于伤后0(即刻)、3、7、10、14 d(注射日注射后)测量创面面积并计算伤后3、7、10、14 d创面愈合率。伤后3、7 d行当日注射后分别处死各组剩余大鼠中的9只,取创周皮肤组织,采用实时荧光定量反转录PCR法检测伤后3 d肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-10及伤后7 dⅠ、Ⅲ型胶原的mRNA表达,采用酶联免疫吸附测定法检测伤后7 d血管内皮生长因子(VEGF)的表达水平,采用蛋白质印迹法检测伤后3 d核因子κB-p65蛋白表达、伤后7 d蛋白激酶B(Akt)的磷酸化水平。对数据行重复测量方差分析、单因素方差分析、LSD-t检验及Bonferroni校正。结果培养24 h,转染HGF的ADSC形态良好,与未转染ADSC无差异,转染率达90%。伤后3、7、10、14 d,HGF修饰ADSC组大鼠创面愈合率分别为(31.5±1.0)%、(75.2±2.0)%、(92.2±1.3)%、(99.1±1.8)%,显著高于PBS组的(21.4±1.3)%、(61.4±1.5)%、
Objective To investigate the effects and mechanism of hepatocyte growth factor(HGF)-modified human adipose mesenchymal stem cells(ADSCs)on the wound healing of full-thickness skin defects in diabetic rats.Methods The experimental research method was adopted.The discarded abdominal adipose tissue was collected from a 35-year-old healthy female who underwent abdominal liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University in December 2019.The long spindle-shaped primary ADSCs were obtained by collagenase digestion,and the third passage of cells were identified by flow cytometry to positively express ADSCs surface markers CD29 and CD90 and negatively express CD34 and CD45.The third passage of ADSCs were used for the subsequent experiments.ADSCs were transfected with lentivirus-mediated HGF for 4 h(obtaining HGF modified ADSCs)and then routinely cultured for 24 h.The cell morphology was observed under an inverted phase contrast microscope,and the transfection rate was calculated.Eighty-one male Sprague-Dawley rats aged 4 weeks were induced into diabetic rat model by high glucose and high fat diet combined with streptozotocin injection.A full-thickness skin defect wound of 1.5 cm×1.5 cm was made on the back of each rat.The injured rats were divided into phosphate buffer solution(PBS)group,ADSCs alone group,and HGF-modified ADSCs group according to the random number table,with 27 rats in each group.The rats were injected with the same volume of corresponding substances around the wound on post injury day(PID)1,3,and 7,respectively.Nine rats in each group were selected according to the random number table,the wound area of whom was measured on PID 0(immediately),3,7,10,and 14(after injection on injection day),and the wound healing rates on PID 3,7,10,and 14 were calculated.Nine remaining rats in each group were sacrificed after injection on PID 3 and 7,respectively,and the skin tissue around the wound were collected.The mRNA expressions of inflammatory factors
作者
张月
韩飞
何亭
计鹏
张智
陶克
Zhang Yue;Han Fei;He Ting;Ji Peng;Zhang Zhi;Tao Ke
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2021年第9期860-868,共9页
Chinese Journal of Burns
基金
国家自然科学基金面上项目(81871561)
国家自然科学基金青年科学基金(82002039)。
关键词
糖尿病
伤口愈合
肝细胞生长因子
脂肪间充质干细胞
慢性创面
炎症反应
Diabetes mellitus
Wound healing
Hepatocyte growth factor
Adipose mesenchymal stem cells
Chronic wounds
Inflammatory response
