瞬时受体电位香草酸亚型4特异性激活剂对人血管内皮细胞功能和大鼠穿支皮瓣血供的影响及其机制  

Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism

作　　者：邝依敏 黄昕[1] 蒙旭昌[2] 顾舒晨 张泽伟 刘云菡 骆申英 昝涛[1] Yimin Khoong;Huang Xin;Meng Xuchang;Gu Shuchen;Zhang Zewei;Liu Yunhan;Luo Shenying;Zan Tao(Department of Plastic and Reconstructive Surgery,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;Department of Plastic and Cosmetic Surgery,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China;Department of Plastic and Reconstructive Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]上海交通大学医学院附属第九人民医院整复外科,上海200011 [2]广西医科大学第一附属医院整形美容外科,南宁530021 [3]郑州大学第一附属医院整形外科,郑州450052

出　　处：《中华烧伤与创面修复杂志》2022年第5期434-446,共13页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(81772086,82072177);上海市“医苑新星”青年医学人才培养资助计划;上海交通大学“晨星-优秀青年学者奖励计划”副教授支持计划A类;上海交通大学医学院“双百人”计划。

摘　　要：目的分析瞬时受体电位香草酸亚型4(TRPV4)激活对人脐静脉内皮细胞(HUVEC)功能及内皮-间质转化(EndMT)的作用,并探讨TRPV4激活对大鼠穿支皮瓣血流灌注和成活的影响及其机制。方法采用实验研究方法。取第3~6代HUVEC进行实验,分为0.5μmol/L 4α-佛波醇12,13-二癸酸酯(4αPDD)组、1.0μmol/L 4αPDD组、3.0μmol/L 4αPDD组、10.0μmol/L 4αPDD组、磷酸盐缓冲液(PBS)组,分别采用相应终物质的量浓度的4αPDD及PBS培养,采用细胞计数试剂盒8(CCK-8)法检测培养6、12 h细胞增殖活性。另取细胞分为PBS组、1μmol/L 4αPDD组、3μmol/L 4αPDD组,同前处理并检测培养6、12、24、48 h的细胞增殖活性;采用划痕试验检测划痕后12、24、48 h剩余划痕面积,并计算剩余划痕面积百分比;采用Transwell实验检测培养24、48 h细胞迁移数量;采用成管实验检测培养4、8 h管状结构数量;采用蛋白质印迹法检测培养24 h细胞上皮钙黏素、神经钙黏素、Slug、Snail蛋白表达。体外实验每组各时间点样本数均为3。取36只8~10周龄雄性SD大鼠,按随机数字表法分为皮瓣延迟组、4αPDD组和生理盐水组,每组12只,均建立背部髂腰动脉穿支皮瓣模型。皮瓣延迟组大鼠仅于皮瓣移植术前1周行皮瓣延迟。4αPDD组和生理盐水组大鼠不行皮瓣延迟,在术前10 min及术后24、48 h,分别经腹腔注射4αPDD和等量的生理盐水。分别于术后0(即刻)、1、4、7 d,行大体观察并计算术后7 d的皮瓣成活率;于术后1、4、7 d,采用激光散斑对比成像技术检测皮瓣血流灌注;术后7 d,采用免疫组织化学染色法检测皮瓣闭塞血管区域微血管密度。体内实验每组各时间点样本数均为12。对数据行析因设计方差分析、重复测量方差分析、单因素方差分析、LSD-t检验、Bonferroni校正。结果培养6、12 h,PBS组、0.5μmol/L 4αPDD组、1.0μmol/L 4αPDD组、3.0μmol/L 4αPDD组、10.0μmol/L 4αPDD组细胞增殖活性�Objective To analyze the effects of transient receptor potential vanilloid type 4(TRPV4)activation on the function and endothelial-to-mesenchymal transition(EndMT)of human umbilical vein endothelial cells(HUVECs),as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism.Methods The experimental research methods were used.The 3rd to 6th passages of HUVECs were used for experiments and divided into 0.5μmol/L 4α-phorbol 12,13-didecanoate(4αPDD)group,1.0μmol/L 4αPDD group,3.0μmol/L 4αPDD group,10.0μmol/L 4αPDD group,and phosphate buffer solution(PBS)group,which were cultivated in corresponding final molarity of 4αPDD and PBS,respectively.The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8(CCK-8).Another batch of cells was acquired and divided into PBS group,1μmol/L 4αPDD group,and 3μmol/L 4αPDD group,which were treated similarly as described before and then detected for cell proliferation activity at 6,12,24,and 48 h of culture.The residual scratch area of cells at post scratch hour(PSH)12,24,and 48 was detected by scratch test,and the percentage of the residual scratch area was calculated.The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment.The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture.The protein expressions of E-cadherin,N-cadherin,Slug,and Snail at 24 h of culture were detected by Western blotting.All the sample numbers in each group at each time point in vitro experiments were 3.A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group,4αPDD group,and normal saline group according to the random number table,with 12 rats in each group,and iliolumbar artery perforator flap models on the back were constructed.The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery.Neither rats in 4αPDD g

关 键 词：TRPV阳离子通道 内皮细胞 穿支皮瓣 新生血管化 病理性 内皮-间质转化 

分 类 号：R622[医药卫生—整形外科]