创面siRNA敲降HO-1改善小鼠放创复合伤创面愈合的实验研究  

Topical knockdown of HO-1 through siRNA improves skin wound healing in mice with radiation-wound combined injury

作　　者：吕晓凡 汪国建 赵娜[2] 龙爽[1] 王双[3] 冉新泽[1] 王军平[4] 王涛[1] LYU Xiaofan;WANG Guojian;ZHAO Na;LONG Shuang;WANG Shuang;RAN Xinze;WANG Junping;WANG Tao(State Key Laboratory of Trauma and Chemical Poisoning,Department of Anti-Radiation Medicine,Institute of Combined Injury,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,40003;Department of Pharmacoutics,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,40003;Biological Science Research Center,Academy for Advanced Interdisciplinary Studies,Southwest University,Chongqing,400715,China;Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038)

机构地区：[1]陆军军医大学(第三军医大学):军事预防医学系防原医学教研室,全军复合伤研究所,创伤与化学中毒全国重点实验室,重庆400038 [2]陆军军医大学(第三军医大学)药学与检验医学系药剂学教研室,重庆400038 [3]西南大学前沿交叉学科研究院生物学研究中心,重庆400715 [4]陆军军医大学(第三军医大学):军事预防医学系,重庆400038

出　　处：《陆军军医大学学报》2024年第11期1194-1205,共12页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82172219)。

摘　　要：目的检测血红素加氧酶1(heme oxygenase-1,HO-1)在放创复合伤(radiation-wound combined injury,R-W-CI)创面修复中的表达情况,评价通过siRNA敲降HO-1对放创复合伤创面愈合的改善作用。方法将36只8周龄雄性C57BL/6J小鼠按随机数字表法分为单纯皮肤创伤组(W组,n=18)和合并全身辐射(6 Gy)损伤的皮肤创伤组(R-W-CI组,n=18),建立单纯皮肤创伤与放创复合伤的小鼠模型。在创面愈合过程中,拍照记录创面愈合情况并通过Image J量化分析残留面积;取材创面组织进行HE染色及病理组织学观察;动态检测外周血象评估造血系统损伤情况。通过创面组织定量PCR及Western blot检测创面HO-1表达水平及变化情况。将26只8周龄雄性C57BL/6J小鼠随机分为siRNA敲降HO-1组(si-HO-1组,n=13)和siRNA阴性对照组(si-NC组,n=13)。放创复合伤致伤后,si-HO-1组在每个创面涂抹负载si-HO-1(5μmol/L)的F127凝胶60μL,si-NC组创面涂抹等量负载阴性对照si-NC的F127凝胶。通过创面组织Western blot检测HO-1的敲降情况,观察创面面积变化,对伤后第3天样本进行定量PCR检测IL-1β、IL-6、TNF-α等炎症因子的表达变化,组织切片进行Ki67免疫组织化学和HE染色;对第9天创面组织进行HE染色病理评估;综合评价敲降HO-1对放创复合伤创面愈合的改善作用。结果与W组相比,创面残留面积的半定量分析表明R-W-CI组愈合在伤后第7、10天显著延迟(P<0.01);第7天的HE病理显示R-W-CI组再上皮化延缓,肉芽组织生长不良;同时R-W-CI组外周血白细胞及其分类计数显示在损伤后早期即显著下降(P<0.05)。检测发现,R-W-CI组创面HO-1蛋白在伤后第3、7天表达略高于W组,但无显著差异(P>0.05),而在第10天显著升高(P<0.05),同时伴有全长与截短形式的分布改变;定量PCR显示R-W-CI组在伤后第7天、10天的创面组织HO-1的表达显著高于W组(P<0.05)。放创复合伤创面siRNA干预实验显示:与si-NC组比较,si-HO-1组能有�Objective To detect the expression profile of heme oxygenase-1(HO-1)during the process of wound repair in radiation-wound combined injury(R-W-CI),and evaluate its wound healing improving effects of R-W-CI by HO-1 knockdown with siRNA.Methods A total of 36 male C57BL/6J mice(8 weeks old)were randomly and equally divided into a simple skin wound group(W group)and a skin wound group combined with whole-body radiation(6 Gy)injury(R-W-CI group).During the wound healing process,the wounds were photographed and recorded,and the residual areas were quantified by Image J.Wound tissues were sampled and stained with HE staining for pathological and histological observation,and the damage to the hematopoietic system was assessed by dynamic examination of the peripheral blood.The expression and changes of HO-1 in wound tissues were detected by q-PCR and Western blotting.Then,26 male C57BL/6J mice(8 weeks old)were randomly and equally divided into siRNA knockdown HO-1 group(si-HO-1 group)and siRNA negative control group(si-NC group).After radiation combined injury was inflicted,60μL of F127 gel loaded with si-HO-1(5μm/L)was applied to each wound in the si-HO-1 group,and an equal amount of F127 gel loaded with negative control si-NC was applied to the wound in the si-NC group.The knockdown of HO-1 in wound tissues was detected by Western blotting,and the changes in wound area were observed.In the wound tissues harvested in 3 d after wounding,the expression of cytokines IL-1β,IL-6 and TNF-αwas examined by q-PCR and the proliferation of granulation tissues was evaluated by Ki67 immunohistochemical staining.HE staining was performed on wound tissues on day 3 and day 9 post-injury to assess the improvement effect of knockdown of HO-1 on wound healing of radiation combined injuries.Results Compared with the W group,semi-quantitative analysis of the residual wound area showed that healing was significantly delayed in the R-W-CI group on days 7 and 10 post-injury(P<0.01).HE staining on day 7 showed that in the R-W-CI group,the re-

关 键 词：放创复合伤 创面愈合 炎症反应 血红素加氧酶1 小干扰RNA 

分 类 号：R394.6[医药卫生—医学遗传学] R64[医药卫生—基础医学] R818.74