含人脂肪来源蛋白复合物的三维生物打印墨水的创面修复效应初探  

Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes

作　　者：张超[1] 李曌[1] 宋薇 姚斌[1] 恩和吉日嘎拉 张孟德 梁莉婷 姜玉峰 付小兵[1] 黄沙[1] Zhang Chao;Li Zhao;Song Wei;Yao Bin;Enhejirigala null;Zhang Mengde;Liang Liting;Jiang Yufeng;Fu Xiaobing;Huang Sha(Research Center for Wound Repair and Tissue Regeneration,Medical Innovation Research Department,the PLA General Hospital,Beijing 100048,China;Department for Wound Repair and Plastic Surgery,PLA Strategic Support Force Characteristic Medical Center,Beijing 100005,China)

机构地区：[1]解放军总医院医学创新研究部创伤修复与组织再生研究中心,北京100048 [2]解放军战略支援部队特色医学中心创面修复整形科,北京100005

出　　处：《中华烧伤杂志》2021年第11期1011-1023,共13页Chinese Journal of Burns

基　　金：国家自然科学基金创新研究群体科学基金项目(81721092);国家自然科学基金重点项目(81830064);国家自然科学基金青年科学基金项目(32000969,82002056);中国医学科学院医学与健康科技创新工程项目(2019-I2M-5-059);解放军总医院军事医学创新研究项目(CX19026);王正国创伤医学发展基金会生长因子复兴计划(SZYZ-TR-03)。

摘　　要：目的探讨人脂肪来源蛋白复合物(ADPC)对人皮肤成纤维细胞(HSF)和人脐静脉内皮细胞(HUVEC)增殖和迁移能力的影响,以及含ADPC的三维生物打印墨水(Bioink)在裸鼠全层皮肤缺损创面中的修复效应。方法采用实验研究方法。收集解放军总医院2020年10月—2021年3月收治的3例行腹部皮瓣转移修补术的女性慢性创面患者(年龄29~34岁)的废弃皮下脂肪组织和同期收治的3名行腹部抽脂术的健康女性(年龄24~36岁)的废弃抽脂术脂肪组织,分别制备正常ADPC(nADPC)及抽脂术ADPC(lADPC)。采用二辛丁酸法测定2种ADPC的蛋白浓度,计算2种ADPC的提取效率,样本数均为3。取对数生长期HSF和HUVEC进行后续实验。取2种细胞均按随机数字表法(分组方法下同)分为磷酸盐缓冲液(PBS)对照组、4μg/mL nADPC组、20μg/mL nADPC组、100μg/mL nADPC组和200μg/mL nADPC组,每组5孔。PBS对照组细胞采用PBS培养,余4组分别采用含相应终质量浓度的nADPC培养。常规培养24 h,采用细胞计数试剂盒8法检测细胞增殖活力。取HSF和HUVEC,分为PBS对照组、单纯nADPC组、单纯lADPC组、单纯肿瘤坏死因子α(TNF-α)组、TNF-α+nADPC组、TNF-α+lADPC组。PBS对照组与单纯TNF-α组细胞分别加入PBS,单纯nADPC组、单纯lADPC组、TNF-α+nADPC组及TNF-α+lADPC组中分别加入终质量浓度100μg/mL的nADPC或lADPC,单纯TNF-α组、TNF-α+nADPC组及TNF-α+lADPC组再加入终质量浓度20 ng/mL的TNF-α。进行细胞划痕试验后计算划痕后24 h细胞迁移率(样本数为3),同前检测培养24 h细胞增殖活力(样本数为5)。取明胶-海藻酸钠复合Bioink(Bioink AG),制备含100μg/mL lADPC的Bioink AG(lADPC-Bioink AG),观察二者在室温下及冷凝后的形态和三维生物打印且交联后的形态,采用流变仪检测流变性能时记录低温成胶时间(样本数为3)。取20只8~10周龄雌性BALB/c-NU裸鼠,建立背部全层皮肤缺损创面模型后,分为常规换药组、单纯lADPC�Objective To investigate the effects of human adipose-derived protein complex(ADPC)on the proliferation and migration ability of human skin fibroblasts(HSFs)and human umbilical vein endothelial cells(HUVECs),and the repairing effects of ADPC-containing three-dimensional(3D)bioprinting ink(Bioink)in full-thickness skin defect wounds of nude mice.Methods The experimental research method was used.Discarded subcutaneous adipose tissue from 3 female patients with chronic wounds(aged 29-34 years)admitted to PLA General Hospital for abdominal flap transfer from October 2020 to March 2021 and discarded liposuction adipose tissue from 3 healthy female(aged 24-36 years)for abdominal liposuction during the same period were collected to prepare normal ADPC(nADPC)and liposuction-derived ADPC(lADPC),respectively.The protein concentration of the two kinds of ADPC was measured by bicinchoninic acid method,and the extraction efficiency of them was calculated.The sample numbers were 3.HSFs and HUVECs in logarithmic growth phase were taken for the subsequent experiments.HSFs and HUVECs were divided into phosphate buffered saline(PBS)control group,4μg/mL nADPC group,20μg/mL nADPC group,100μg/mL nADPC group,and 200μg/mL nADPC group according to the random number table(the same grouping method below),with 5 wells in each group.Cells in PBS control group were cultured with PBS,and the cells in the 4 remaining groups were cultured with the corresponding final mass concentration of nADPC.After 24 h of conventional culture,the cell proliferation viability was detected by cell counting kit 8 method.HSFs and HUVECs were taken and divided into PBS control group,nADPC alone group,lADPC alone group,tumor necrosis factor-α(TNF-α)alone group,TNF-α+nADPC group,and TNF-α+lADPC group.Cells in PBS control group and TNF-αalone group were added with PBS.nADPC or lADPC was added to the cells in nADPC alone group,lADPC alone group,TNF-α+nADPC group,and TNF-α+lADPC group with a final mass concentration of 100μg/mL,respectively.TNF-αwith a fi

关 键 词：再生医学 组织工程 多蛋白复合物 三维生物打印墨水 创面修复 

分 类 号：R64[医药卫生—外科学] R605[医药卫生—临床医学]