低氧预处理大鼠脂肪源性间充质干细胞条件培养基对大鼠全层皮肤缺损创面愈合的影响  

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

作　　者：郜敏[1] 张杰[1] 王际壮 刘琰[1] 章雄[1] 施燕[1] Gao Min;Zhang Jie;Wang Jizhuang;Liu Yan;Zhang Xiong;Shi Yan(Department of Burns and Plastic Surgery,Ruijin Hospital,Shanghai JiaoTong University School of Medicine,Shanghai 200001,China)

机构地区：[1]上海交通大学医学院附属瑞金医院烧伤整形科,200001

出　　处：《中华烧伤杂志》2020年第9期803-812,共10页Chinese Journal of Burns

基　　金：国家自然科学基金(81871564);上海市科学技术委员会自然科学基金(18ZR1423800);上海市临床重点专科项目(shslczdzk02302)。

摘　　要：目的探讨低氧预处理大鼠脂肪源性间充质干细胞(ADSC)条件培养基对全层皮肤缺损大鼠创面愈合的影响。方法(1)取6周龄雄性SD大鼠1只,颈椎脱臼处死,分离双侧腹股沟脂肪组织,胶原酶消化法提取第3代ADSC,观察细胞形态后用于后续实验。取细胞,采用随机数字表法(分组方法下同)分为成脂诱导组和成骨诱导组,每组各6孔。成脂诱导组培养14 d观察成脂情况,成骨诱导组培养28 d观察成骨情况。(2)取第3代ADSC,分为常氧组和低氧组,常氧组细胞置于氧气体积分数20%的常氧培养箱中培养,低氧组细胞置于氧气体积分数2%的低氧培养箱中培养。常氧组培养3 h,低氧组培养3、6、12、24、48 h,分别取3个样本,进行后续指标检测。实时荧光定量反转录PCR检测低氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、过氧化物酶体增殖物激活受体γ(PPAR-γ)的mRNA表达量。收集2组细胞培养上清液,离心过滤后获得常氧条件培养基(normo-CM)和低氧条件培养基(hypo-CM),酶联免疫吸附测定法检测条件培养基中VEGF、转化生长因子β(TGF-β)、表皮生长因子(EGF)和胰岛素样生长因子(IGF)含量。(3)取27只6~8周龄雄性SD大鼠,分为磷酸盐缓冲液(PBS)组、normo-CM组和hypo-CM组,每组9只,在其背部制作直径为1 cm的圆形全层皮肤缺损创面并分别滴加50μL PBS、normo-CM及hypo-CM。伤后0、3、5、7、9、11 d,观察创面大体情况,测量创面面积并计算创面未愈合率。取创面组织,苏木精-伊红染色观察伤后3、9、11 d创面炎症反应及伤后9 d创面再上皮化水平;Masson染色观察伤后11 d创面胶原沉积情况,并分析胶原容积分数(CVF)。对数据行重复测量方差分析、单因素方差分析、t检验、Bonferroni校正。结果(1)细胞融合度低时呈长梭形、贴壁生长、排列紧密。培养14 d,成脂诱导组细胞经油红O染色后被染成红色的脂滴Objective To investigate the effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells(ADSCs)conditioned medium on wound healing of rats with full-thickness defects.Methods(1)A 6-week-old male Sprague-Dawley rat was sacrificed by cervical dislocation,the bilateral inguinal adipose tissue was collected,the third generation ADSCs were isolated by collagenase digestion method,and the cells morphology was observed.The cells were harvested and divided into adipogenic induction group and osteogenic induction group according to the random number table(the same grouping method below),with 6 wells in each group.The cells in adipogenic induction group were cultured for 14 days to observe adipogenesis,and cells in osteogenic induction group were cultured for 28 days to observe osteogenesis.(2)The third generation ADSCs were collected and divided into normoxic group and hypoxic group.Cells in normoxic group was incubated in normal oxygen incubator with oxygen volume fraction of 21%,and cells in hypoxic group was incubated in low oxygen incubator with oxygen volume fraction of 2%respectively,with 3 samples in each group for each time point.Three samples in normoxic group on 3 h of culture and in hypoxic group on 3,6,12,24,and 48 h of culture were collected for detecting the following indexes.The mRNA expressions of hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF),and peroxisome proliferator-activated receptorγ(PPAR-γ)were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction.The cell culture supernatant in the two groups was collected,centrifuged,and filtered to obtain normoxic conditioned medium(normo-CM)and hypoxic conditioned medium(hypo-CM).Enzyme linked immunosorbent assay was used to detect content of VEGF,transforming growth factorβ(TGF-β),epidermal growth factor(EGF),and insulin-like growth factor(IGF)in conditioned medium.(3)Twenty-seven male Sprague-Dawley rats aged 6-8 weeks were collected

关 键 词：伤口愈合 缺氧 脂肪源性间充质干细胞 条件培养基 

分 类 号：R644[医药卫生—外科学] Q493[医药卫生—临床医学]