重组人粒细胞巨噬细胞集落刺激因子对糖尿病大鼠创面愈合及微小RNA表达的影响
중조인립세포거서세포집락자격인자대당뇨병대서창면유합급미소RNA표체적영향
Effects of recombinant human granulocyte-macrophage colony-stimulating factor on wound healing and microRNA expression in diabetic rats
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2014年 3期 243-250 ,共8页

    刘移峰%刘德伍%郭光华%毛远桂%汪显林

    류이봉%류덕오%곽광화%모원계%왕현림

    粒细胞巨噬细胞集落刺激因子,重组%糖尿病,实验性%皮肤%伤口愈合%基因表达谱%微小RNA 粒細胞巨噬細胞集落刺激因子,重組%糖尿病,實驗性%皮膚%傷口愈閤%基因錶達譜%微小RNA
    립세포거서세포집락자격인자,중조%당뇨병,실험성%피부%상구유합%기인표체보%미소RNA
    Granulocyte macrophage colony-stimulating factors,recombinant%Diabetes mellitus,experimental%Skin%Wound healing%Gene expression profiling%MicroRNA
    目的 探讨重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)凝胶对糖尿病大鼠创面愈合及创面组织中微小RNA表达的影响. 方法 取18只雄性清洁级SD大鼠建立糖尿病模型.4周后取稳定成模的16只糖尿病大鼠,于背部两侧制作4个全层皮肤缺损创面,共64个创面.采用配对设计以脊柱两侧对称的创面配对,采用盲法(用红蓝两色标识rhGM-CSF或凝胶基质)及随机数字表法分为2组,每组32个创面;A组采用红色标签药剂、B组采用蓝色标签药剂.2组大鼠均每日涂抹药膏1次,厚度为3 mm,至伤后14d.伤后3、7、14d,大体观察各组创面愈合情况;计算伤后3、7d创面愈合率.伤后3、7、14d,每组分别采集2、4、8个创面标本行HE染色观察组织病理学改变,免疫组织化学染色检测创面CD31和增殖细胞核抗原(PCNA)表达(数据以吸光度值表示).伤后7d每组另采集6个创面组织样本行芯片检测,筛选差异表达明显的微小RNA,经实时荧光定量RT-PCR验证后行京都基因和基因组百科全书(KEGG)信号通路功能富集分析.对数据行配对样本t检验或两样本t检验. 结果 (1)伤后3d,2组创面面积均明显缩小,肉芽组织增生明显.伤后7d,2组创面均进一步缩小,创腔基本被肉芽组织填平;A组创面缩小更明显,肉芽组织量多.伤后14d,A组创面基本愈合;B组部分创面仍存在少许创腔及结痂.伤后3、7d,A组创面愈合率分别为(41±5)％和(75±4)％,明显高于B组的(31±9)％和(71±4)％(t值分别为10.13、8.06,P值均小于0.001).(2)伤后3d,2组创面表皮细胞、内皮细胞、Fb均排列稀疏,炎性细胞大量浸润.其中A组创面上述表现优于B组.伤后7d,A组创面表皮细胞、内皮细胞、Fb排列渐致密,炎性细胞浸润稍多于B组.伤后14 d,A组表皮完全覆盖创面,B组部分创面仍有炎性浸润.(3)伤后3、7、14 d,A组创面CD31和PCNA阳性表达(0.275 ±0.018、0.345±0.034、0.305±0.023,0.406±0.063、0.223±0.011、0.045±0.022)均较B组(0.222±0.020、0.229 ±0.018、0.197 ±0.015,0.324 ±0.039、0.162±0.012、0.018 ±0.020)明显(t值为2.281～9.652,P＜0.05或P＜0.01).(4)微小RNA芯片检测筛选,与B组比较,A组创面组织中上调的微小RNA有18个、下调的有13个.(5)实时荧光定量RT-PCR的验证结果与芯片检测结果较一致.(6)KEGG信号通路功能富集分析显示31个差异表达明显的微小RNA中有4个参与MAPK信号途径,3个参与Wnt信号途径,1个参与TGF-β信号途径,3个参与表皮生长因子受体信号途径,2个参与细胞周期途径,5个参与轴突导向信号途径,6个参与黏着斑途径,3个参与肌动蛋白细胞骨架途径,1个参与细胞外基质受体途径,3个参与黏着连接途径,1个参与细胞黏附分子途径.经揭肓,A组采用红色标签药剂为rhGM-CSF凝胶、B组采用蓝色标签药剂为凝胶基质. 结论 rhGM-CSF凝胶可促进糖尿病大鼠创面愈合,引起皮肤组织中微小RNA明显差异性表达,这可能是其促进创面愈合在基因转录后水平上的靶点.
    목적 탐토중조인립세포거서세포집락자격인자(rhGM-CSF)응효대당뇨병대서창면유합급창면조직중미소RNA표체적영향. 방법 취18지웅성청길급SD대서건립당뇨병모형.4주후취은정성모적16지당뇨병대서,우배부량측제작4개전층피부결손창면,공64개창면.채용배대설계이척주량측대칭적창면배대,채용맹법(용홍람량색표식rhGM-CSF혹응효기질)급수궤수자표법분위2조,매조32개창면;A조채용홍색표첨약제、B조채용람색표첨약제.2조대서균매일도말약고1차,후도위3 mm,지상후14d.상후3、7、14d,대체관찰각조창면유합정황;계산상후3、7d창면유합솔.상후3、7、14d,매조분별채집2、4、8개창면표본행HE염색관찰조직병이학개변,면역조직화학염색검측창면CD31화증식세포핵항원(PCNA)표체(수거이흡광도치표시).상후7d매조령채집6개창면조직양본행심편검측,사선차이표체명현적미소RNA,경실시형광정량RT-PCR험증후행경도기인화기인조백과전서(KEGG)신호통로공능부집분석.대수거행배대양본t검험혹량양본t검험. 결과 (1)상후3d,2조창면면적균명현축소,육아조직증생명현.상후7d,2조창면균진일보축소,창강기본피육아조직전평;A조창면축소경명현,육아조직량다.상후14d,A조창면기본유합;B조부분창면잉존재소허창강급결가.상후3、7d,A조창면유합솔분별위(41±5)％화(75±4)％,명현고우B조적(31±9)％화(71±4)％(t치분별위10.13、8.06,P치균소우0.001).(2)상후3d,2조창면표피세포、내피세포、Fb균배렬희소,염성세포대량침윤.기중A조창면상술표현우우B조.상후7d,A조창면표피세포、내피세포、Fb배렬점치밀,염성세포침윤초다우B조.상후14 d,A조표피완전복개창면,B조부분창면잉유염성침윤.(3)상후3、7、14 d,A조창면CD31화PCNA양성표체(0.275 ±0.018、0.345±0.034、0.305±0.023,0.406±0.063、0.223±0.011、0.045±0.022)균교B조(0.222±0.020、0.229 ±0.018、0.197 ±0.015,0.324 ±0.039、0.162±0.012、0.018 ±0.020)명현(t치위2.281～9.652,P＜0.05혹P＜0.01).(4)미소RNA심편검측사선,여B조비교,A조창면조직중상조적미소RNA유18개、하조적유13개.(5)실시형광정량RT-PCR적험증결과여심편검측결과교일치.(6)KEGG신호통로공능부집분석현시31개차이표체명현적미소RNA중유4개삼여MAPK신호도경,3개삼여Wnt신호도경,1개삼여TGF-β신호도경,3개삼여표피생장인자수체신호도경,2개삼여세포주기도경,5개삼여축돌도향신호도경,6개삼여점착반도경,3개삼여기동단백세포골가도경,1개삼여세포외기질수체도경,3개삼여점착련접도경,1개삼여세포점부분자도경.경게황,A조채용홍색표첨약제위rhGM-CSF응효、B조채용람색표첨약제위응효기질. 결론 rhGM-CSF응효가촉진당뇨병대서창면유합,인기피부조직중미소RNA명현차이성표체,저가능시기촉진창면유합재기인전록후수평상적파점.
    Objective To investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats.Methods Eighteen male SD rats of clean grade were used to reproduce diabetes model.Four weeks later,a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes,with 4 wounds on each rat.Two symmetrical wounds on either side of the spine were created as a pair according to paired design.Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle),with 32 wounds in each group.The ointment with red tag was applied on the wounds of group A and the blue one on group B.The application was conducted once a day,with a thickness of 3 mm,up to post injury day (PID) 14.Gross observation of wound healing was conducted on PID 3,7,14.The wound healing rate was determined on PID 3 and 7.On PID 3,7,14,tissues from 2,4,and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining,and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value).On PID 7,tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs.Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG)signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR.Data were processed with paired t test or two-sample t test.Results (1) On PID 3,the wound area was significantly decreased,and the wound granulation was significantly proliferated in both groups.On PID 7,the wound area was further decreased,and the wound area was almost filled by granulation in both groups; the conditions in group A were better.On PID 14,all the wounds in group A were almost healed,while a small area of raw wound with incrustation still remained in some wounds of group B.On PID 3 and 7,the wound healing rates of group A were (41 ± 5)％ and (75 ±4)％,significantly higher than those of group B [(31 ±9)％ and (71 ±4)％,withtvalues respectively 10.13 and 8.06,P values below 0.001].(2) On PID 3,the epidermal cells,endothelial cells,and Fbs in the wounds of 2 groups were sparse,with heavy infiltration of inflammatory cells.The above condition in the wounds was better in group A than in group B.On PID 7,the epidermal cells,endothelial cells,and Fbs were gradually well arranged in group A; infiltration of inflammatory cells decreased,and the condition was better than that of group B.On PID 14,the wounds of group A were completely covered by epidermis,while infiltration of inflammatory cells still remained in some wounds of group B.(3) On PID 3,7,14,the positive expressions of CD31 and PCNA in group A were respectively 0.275 ± 0.018,0.345 ±0.034,0.305 ±0.023; 0.406 ±0.063,0.223±0.011,0.045 ±0.022.They were significantly higher than those of group B (0.222 ±0.020,0.229 ±0.018,0.197 ±0.015; 0.324 ±0.039,0.162 ± 0.012,0.018 ±0.020,with t values from 2.281 to9.652,P ＜0.05 orP ＜0.01).(4) According to the microRNAs detection and screening,as compared with group B,18 microRNAs were up-regulated while 13 were down-regulated in the wounds of group A.(5) The results of real-time fluorescent quantitative RT-PCR had good consistency with the results of microRNAs detection.(6) Enrichment analysis of KEGG signaling pathway showed that among the 31 differentially expressed microRNAs,4 took part in the MAPK signaling pathway,3 took part in the Wnt signaling pathway,1 took part in the TGF-β signaling pathway,3 took part in the epidermal growth factor receptor signaling pathway,2 took part in the cell cycle pathway,5 took part in the axon guidance signaling pathway,6 took part in the focal adhesion pathway,3 took part in the regulation of actin cytoskeleton pathway,1 took part in the extracellular cell matrix receptor pathway,3 took part in the adherens junction pathway,and 1 took part in the cell adhesion molecules pathway.After disclosing the blind,it showed that the ointment with red tag was the rhGM-CSF gel and the blue one was gel vehicle.Conclusions The rhGM-CSF gel can pronote wound healing in diabetic rats,producing significant differential microRNA expression in wounds,and they may be the target at gene post-transcriptional level of rhGM-CSF gel in promoting wound healing.
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