贝伐珠单抗注射液对食蟹猴皮肤伤口愈合的影响_杨平.pdf

DOI:10.12300/j.issn.1674-5817.2022.128实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)崔 立，上海交通大学农业与生物学院动物科学系研究员、博士生导师、比较医学学科组PI。研究涉及肠道微生物与宿主互作、病毒宏基因组学等。近年主要关注疾病动物模型，从事基于肠炎模型和类器官探究黏膜损伤修复机制，并进行黏膜修复活性物质的评估等研究工作。同时担任上海交通大学实验动物伦理委员会委员，中国畜牧兽医学会公共卫生分会第一、第二届理事，中国实验动物学报中国比较医学杂志亚洲兽医案例分析等杂志编委，英国皇家兽医学院访问学者。主持完成国家级及上海市科研项目10余项，主编上海及国家职业培训系列教材宠物健康护理员，发表学术论文40余篇，获得授权专利8项。杨 平，上海交通大学农业与生物学院动物医学在读硕士研究生，中国执业兽医师。从事自身免疫疾病和神经系统疾病的临床前药物药效评价服务工作已13年，作为项目负责人多次服务制药公司或学术研究机构，完成国内外新药临床试验申请（investigational newdrug application，IND）的药效学实验数据申报10多项，参与完成有关动物疾病模型及相关设备的研发项目近10项，获得发明专利4项，其中原始取得3项，继受取得1项，第一发明人1项。贝伐珠单抗注射液对食蟹猴皮肤伤口愈合的影响杨 平1,崔 立1,俞 诚2,温智越11.上海交通大学农业与生物学院,上海 201209;2.澎立生物医药技术(上海)股份有限公司,上海 201203摘要 目的通过观察贝伐珠单抗注射液对食蟹猴背部皮肤伤口愈合速度及损伤皮肤组织中CD34表达的影响，验证贝伐珠单抗注射液对食蟹猴伤口愈合的延迟作用，为临床肿瘤手术患者使用贝伐珠单抗治疗时提供剂量和频次的用药参考。方法6只雄性食蟹猴采用背部全层皮肤切除术建立伤口愈合评估模型，随机选取3只食蟹猴作为生理盐水组，其余3只食蟹猴为贝伐珠单抗组。贝伐珠单抗组食蟹猴在手术后第0、4、8、11天静脉注射30 mg/kg 贝伐珠单抗注射液，生理盐水组静脉注射等量生理盐水；术后进行血常规检测，并观察各组食蟹猴的伤口愈合情况，Image J软件分析伤口愈合率，用评分法对伤口状态的严重程度进行评价。4周后对6只食蟹猴伤口愈合皮肤取材，通过免疫组织化学法检查皮肤组织中CD34的表达水平。结果成功构建食蟹猴伤口愈合评估模型。术后第3天的食蟹猴血液中白细胞、中性粒细胞数量明显升高（P0.05），第7天后白细胞数量逐渐恢复正常，而红细胞、血红蛋白、红细胞压积没有明显变化，提示动物营养状态良好。与生理盐水组相比，贝伐珠单抗组的伤口愈合率在术后7 d、28 d明显降低（P0.01，P0.001），愈合皮肤组织中CD34表达水平明显降低（P0.01）。结论贝伐珠单抗注射给药可能通过抑制食蟹猴伤口新生皮肤组织中的微血管生成，延缓伤口愈合。临床中患者手术后使用贝伐珠单抗注射液时应该权衡利弊，科学合理地选择干预时间点、剂量、频次。关键词 贝伐珠单抗注射液；伤口愈合；伤口评分；CD34；食蟹猴中图分类号 Q95-33；R-332 文献标志码 A 文章编号 1674-5817（2023）01-0021-09Effects of Bevacizumab Injection on the Skin Wound Healing in Cynomolgus MonkeysYANG Ping1,CUI Li1,YU Cheng2,WEN Zhiyue11.Shanghai Jiao Tong University School Of Agriculture And Biology,Shanghai 201209,China;2.PharmaLegacy Laboratories(Shanghai)Co.,Ltd.,Shanghai 201203,ChinaCorrespondence to:CUI Li(ORCID:0000-0001-5202-9671),E-mail: 人类疾病动物模型：药学专题 Animal Model of Human Disease:Pharmacology基金项目 上海市科委科技计划项目-实验动物专项“基于肠类器官3D培养和高内涵成像的药物毒性和安全性评价模型研究”（21140901400）第一作者 杨 平（1986），男，硕士研究生，执业兽医师，研究方向：临床前药物药理药效评价。E-mail：通信作者 崔立（1971），女，博士，研究员，研究方向：人兽共患病与比较医学。E-mail：。ORCID：0000-0001-5202-967121实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)ABSTRACT Objective By observing the impact of Bevacizumab injection on the speed of wound healing and the expression of CD34 in cynomolgus monkeys,to verify its delayed wound healing mechanism,and to provide clinical reference for the dosage and frequency of Bevacizumab treatment for oncology surgery patients.Methods Six male cynomolgus monkeys underwent full-thickness skin resection on the back to establish a wound healing evaluation model.Three cynomolgus monkeys were randomly selected as the saline group,and the other three cynomolgus monkeys were allocated to the Bevacizumab treatment group.The monkeys of Bevacizumab group were administrated with Bevacizumab injection(30 mg/kg,i.v.)on day 0,day 4,day 8,and day 11,respectively.The monkeys of saline group were injected with the same volume of saline as the group receiving Bevacizumab.The blood routine test was conducted,and the wound healing of each group of cynomolgus monkeys was observed 28 days after operation.The wound healing rate was analyzed by software Image J,and the severity of the wound was assessed by scoring method.Wound healing skin samples of 6 cynomolgus monkeys were obtained after 4 weeks,and the expression level of CD34 in the wound skin tissues was detected using immunohistochemistry.Results A skin excision wound model has already been established in cynomolgus monkeys.The number of white blood cells and neutrophils in the blood of cynomolgus monkeys increased significantly on the 3rd day after operation(P0.05),and the number of white blood cells gradually returned to normal on the 7th day,while the number of red blood cells,hemoglobin and hematocrit did not change significantly,suggesting that the animals were in good nutritional status.The Bevacizumab injection group wound healing rate was significantly slower than that of the saline group on day 7,and day 28(P0.01,P0.001).Microvessel density in the wound skin of Bevacizumab group appeared to be significantly lower than that of the saline group(P0.01).ConclusionThe injection of Bevacizumab may delay the wound healing by inhibiting the angiogenesis in the new skin tissue of the cynomolgus monkey.In clinical practice,the timing of giving a tumor patient a bevacizumab injection after surgery should be based on the pros and cons,and the duration,dosage,and frequency of the intervention should be chosen in a rational way.Key words Bevacizumab injection;Wound healing;Wound score;CD34;Cynomolgus monkey贝伐珠单抗是一种抗人血管内皮生长因子全人源化 单 克 隆 抗 体（recombinant humanized anti vascular endothelial growth factor monoclonal antibody，rhuMab VEGF），可 与 游 离 血 管 内 皮 生 长 因 子（vascular endothelial growth factor，VEGF）结合，抑制肿瘤组织新生血管生成，从而发挥抑制肿瘤生长、迁移的作用。现阶段，贝伐珠单抗已被广泛纳入多种恶性肿瘤诊疗指南的全球推荐标准方案，如转移性结直肠癌、非小细胞肺癌、脑神经胶质瘤、肾癌、卵巢癌和宫颈癌等1-5。rhuMab VEGF 在肿瘤治疗中发挥显著作用，但其局限性仍不可忽视，其中就包括伤口愈合延迟。VEGF在伤口的愈合过程中具有促进炎性反应、促进切口部位新生血管生成等作用，而接受贝伐珠单抗治疗的患者伤口愈合延迟，出现手术并发症的概率明显增加6。研究表明，创伤愈合是一个极为复杂的过程，按照其病理变化，可分为止血期、炎性期、增生期及成熟期（重塑期）4个阶段。在伤口愈合过程中，炎性细胞如中性粒细胞及巨噬细