目的 构建一种皮肤感染性慢性溃疡动物模型。 方法 C57BL/6小鼠通过药物处理、磁铁造成创面后，使用3M无菌透明敷料对伤口进行覆盖，观测和记录小鼠伤口的变化。挤压后的第3、7、14、21、28 d，大体拍照，伤口部位取材，通过HE染色、Masson染色、免疫组化染色、16SrDNA检测观察伤口的病理变化。 结果 大体观察第3天开始有腐肉覆盖，渗出液渗出，至28天腐肉增多，第1~28天伤口愈合率均小于0；与正常皮肤相比，HE染色显示，伤口部位存在大量炎症细胞浸润和坏死组织碎片；Masson染色显示，伤口部位仅少量胶原纤维散在分布于坏死组织中，且排列紊乱松散；继而免疫组化显示，伤口部位M1型巨噬细胞阳性率显著高于周围正常皮肤（P<0.001），M2型阳性率没有差异，M1与M2型巨噬细胞的比值最高可达10；免疫组化显示第14天炎症因子TNF-α和IL-1β在伤口部位高表达；进一步16SrDNA检测显示，第0天菌群多样化明显，第3天之后变形菌门占据90%以上，伤口细菌多样化消失，出现菌群失调。 结论 本研究利用药物提高小鼠氧化应激水平，磁铁挤压的方法模拟伤口部位组织缺血坏死，3M膜促进伤口腐肉形成和菌群失调，通过大体观察、组织学染色、免疫组化和菌群16SrDNA结果均符合临床上皮肤慢性溃疡的发展特征。该造模方法可以成功构建一种皮肤感染性慢性溃疡动物模型。
Abstract
Objective To construct an animal model of cutaneous infectious chronic ulcer. Methods After the wound of C57BL/6 mice was caused by drug treatment and magnet, the wound was covered with 3M sterile transparent dressing, and the changes of the mouse wound were observed and recorded. On the 3rd, 7th, 14th, 21st and 28th days after extrusion, the wound site was taken from the material, and the pathological changes of the wound were observed by HE staining, Masson staining, immunohistochemical staining and 16SrDNA detection. Results In general, it was observed that there was carrion covering on the 3rd day, exudate exudation, and the carrion increased on the 28th day, and the wound healing rate was less than 0 on the 1st-28th day. Compared with normal skin, HE staining showed a large number of inflammatory cell infiltrates and necrotic tissue fragments at the wound site; Masson staining showed that only a small number of collagen fibers were scattered in the necrotic tissue at the wound site, and the arrangement was loose; Immunohistochemistry showed that the positive rate of M1 macrophages at the wound site was significantly higher than that of the surrounding normal skin (P<0.001), there was no difference in the positive rate of M2 type, and the ratio of M1 to M2 macrophages was up to 10; immunohistochemistry showed that the inflammatory factors TNF-α and IL-1β were highly expressed at the wound site on day 14; 16SrDNA detection showed that the bacterial diversity on day 0 was obvious, and after the third day, the proteobacteria phylum accounted for more than 90%, and the diversity of wound bacteria disappeared, and dysbacteriosis appeared. Conclusion In this study, the use of drugs to increase the level of oxidative stress in mice, the method of magnet extrusion simulated ischemic necrosis of wound tissues, and the 3M membrane promoted the formation of wound carrion and dysbacteria, and the results of general observation, histological staining, immunohistochemistry and microbiota 16SrDNA are all consistent with the development characteristics of chronic skin ulcers in clinical practice. This modeling method can successfully construct an animal model of skin infectious chronic ulcers.
关键词
皮肤慢性溃疡 /
/
/
炎症 /
/
/
巨噬细胞 /
/
/
菌群失调
Key words
Skin ulcer /
/
/
Inflammation /
/
/
/
Macrophages /
/
/
Dysbiosis
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Clark RA, Ghosh K, Tonnesen MG. Tissue engineering for cutaneous wounds[J]. Invest Dermatol. 2007,127(5):1018-29. DOI: 10.1038/sj.jid.5700715.
[2] Chouhan D, Dey N, Bhardwaj N, et al. Emerging and innovative approaches for wound healing and skin regeneration: Current status and advances[J].Biomaterials.2019,216:119267.DOI:10.1016/j.biomate rials. 2019.119267.
[3] Kim JH, Yang B, Tedesco A, et al. High Levels of Oxidative Stress and Skin Microbiome are Critical for Initiation and Development of Chronic Wounds in Diabetic Mice[J]. Scientific Reports. 2019,9(1):19318. DOI: 10.1038/s41598-019-55644-3.
[4] 徐凝, 吴闽枫, 段彦娟. 慢性皮肤溃疡动物模型的研究进展[J]. 中国中医基础医学杂志, 2020,26(5):704-707. DOI: CNKI:SUN:ZYJC.0.2020-05-047.
[5] 苑海刚, 薛凤, 赵钢. 蓬子菜水溶液对大鼠皮肤溃疡创面修复影响的实验研究[J]. 中医药学报, 2016,44(2):40-42. DOI; CNKI:SUN:ZYXB.0.2016-02-015.
[6] 马忠丽, 张汉庆, 覃剑, 等. 中药“敛疡散”对慢性皮肤溃疡的作用机制研究[J]. 中国中医骨伤科杂志, 2007,15(4):6. DOI: 10.3969/j.issn.1005-0205.2007.04.007.
[7] 王汉峰. 麦卢卡蜂蜜联合银离子敷料对铜绿假单胞菌感染创面及生物膜的影响[D]. 兰州大学, 2021. DOI: 10.27204/d.cnki.glzhu.2021.002901.
[8] 李媛, 赵光明, 董建勋, 等. 中医病证相符的糖尿病慢性皮肤溃疡大鼠造模方法初探[J]. 中国实验动物学报, 2014,22(1):63-66+12. DOI: 10.3969/j.issn.1005-4847.2014.01.012.
[9] Dhall S, Do DC, Garcia M, et al. Generating and reversing chronic wounds in diabetic mice by manipulating wound redox parameters[J]. Journal of diabetes research. 2014,2014:562625. DOI: 10.1155/2014/562625.
[10]Huang N Q, Jin H, Shi J S, et al. Research Progress of Resveratrol Inhibiting Microglial Activation in Neuroinflammation[J]. Chinese Pharmaceutical Journal, 2018,53(2):85-91. DOI: 10.11669/cpj. 2018. 02.001.
[11]Rodrigues M, Kosaric N, Bonham CA, et al. Wound Healing: A Cellular Perspective[J]. Physiological Reviews. 2019,99(1):665-706. DOI: 10.1152/physrev.00067.2017.
[12]刘燕芳, 毕新岭, 陈珏. 糖尿病鼠皮肤伤口模型研究进展[J]. 解放军护理杂志,2017,34(6):45-47. DOI: 10.3969/j.issn.1008-9993. 2017. 06.011.
[13]Schäfer M, Werner S. Oxidative stress in normal and impaired wound repair[J]. Pharmacological research. 2008,58(2):165-71. DOI: 10.1016/j.phrs.2008.06.004.
[14]Kim JH, Martins-Green M. Protocol to Create Chronic Wounds in Diabetic Mice[J]. Journal of Visualized Experiments. 2019,(151). DOI: 10.3791/57656.
[15]Dunning S, Ur Rehman A, Tiebosch MH, et al. Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death[J]. Biochim Biophys Acta. 2013,1832(12):2027-34. DOI: 10.1016/j.bbadis.2013.07.008
[16]叶祝君, 左丹, 张红侠, 等. 人脐带间充质干细胞修复老龄啮齿类动物Ⅲ度压疮的效果研究[J]. 实用老年医学, 2018,32(5):430-432. DOI: CNKI:SUN:SYLA.0.2018-05-008.
[17]Stadler I, Zhang RY, Oskoui P, et al. Development of a simple, noninvasive, clinically relevant model of pressure ulcers in the mouse[J]. Journal of Investigative Surgery. 2004,17(4):221-7. DOI: 10.1080/08941930490472046.
[18]Pegalajar-Jurado A, Easton C D, Crawford R J, et al. Fabrication of a platform to isolate the influences of surface nanotopography from chemistry on bacterial attachment and growth[J]. Biointerphases. 2015,10(1):11002. DOI: 10.1116/1.4913377.
[19]Seth A K, Geringer M R, Hong S J, et al. In vivo modeling of biofilm-infected wounds: A review[J]. Journal of Surgical Research. 2012,178(1)330-338. DOI: 10.1016/j.jss.2012.06.048.
[20]韩晓明, 罗宇慧, 施亚芳等. 慢性皮肤溃疡的疮疡模型治疗试验研究[J]. 中国医学创新, 2008,5(36):1-3. DOI: 10.3969/j.issn.1674-4985.2008.36.001.
[21]Mendes AI, Peixoto MJ, Marques AP, et al. An optimized mouse model of Staphylococcus aureus infected diabetic ulcers[J]. BMC research notes. 2022,15(1):293. DOI: 10.1186/s13104-022-06170-5.
[22]孔遐妮. 3M透明敷料对新生儿一次性电极片造成的接触性皮炎缓解效果[J]. 医学食疗与健康, 2022,20(4):58-60.
[23]Huang Y, Xiao Z, Cao Y, et al. Rapid microbiological diagnosis based on 16S rRNA gene sequencing: A comparison of bacterial composition in diabetic foot infections and contralateral intact skin[J]. Front Microbiol. 2022,13:1021955. DOI: 10.3389/fmicb.2022.1021955.
[24]陈紫璐, 吴金结, 智伟, 等. 藏药十八味党参丸提取物对脂多糖诱导的RAW264.7细胞Akt/p38MAPK信号通路及M1/M2极化影响的探究[J]. 中国药学杂志, 2022,57(01):38-43.
[25]Luca P, Elisabetta G, Denisa B, et al. Macrophage Polarization in Chronic Inflammatory Diseases: Killers or Builders?[J]. Journal of Immunology Research, 2018,2018(12):1-25. DOI: 10.1155/2018/8917804.
[26]Martinez F O, Helming L, Gordon S. Alternative activation of macrophages: an immunologic functional perspective[J]. Annual Review of Immunology, 2009,27(1):451-483. DOI: 10.1146/annurev.immunol.021908.132532.
[27]杨晔琴, 张纯瑜, 孙艳, 等. Ⅲ°压疮皮肤组织iNOS、P53和细胞凋亡的变化[J]. 中国应用生理学杂志, 2011,27(01):50-51+61+133. DOI:10.13459/j.cnki.cjap.2011.01.015.
[28]Fujisaka S, Usui I, Bukhari A, et al. Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice. Diabetes. 2009,58(11):2574-82. DOI: 10.2337/db08-1475.
基金
广东省自然科学基金项目（2019A1515011213, 2020A1515011067,）；广东省医学科学技术研究基金面上项目（A2020136）；新疆维吾尔自治区自然科学基金面上项目（2019D01A83）；广东省药品监督管理局科技创新重点实验室项目(2023ZDZ12)
PDF(10506 KB)
京ICP备08002354号-3
