组织工程化口腔黏膜等效物的血管化特征  

Vascularization characteristics of tissue-engineered oral mucosa equivalents

作　　者：石丽娟 韦建 张旋 何凌霄 江小茜 聂敏海 陈佳娜 刘旭倩 Shi Lijuan;Wei Jian;Zhang Xuan;He Lingxiao;Jiang Xiaoxi;Nie Minhai;Chen Jiana;Liu Xuqian(Department of Periodontal Mucosal Diseases,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Luzhou Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,Luzhou 646000,Sichuan Province,China;Institute of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属口腔医院牙周黏膜病科,四川省泸州市646000 [2]口颌面修复重建和再生泸州市重点实验室,四川省泸州市646000 [3]西南医科大学口腔医学研究所,四川省泸州市646000

出　　处：《中国组织工程研究》2025年第22期4748-4760,共13页Chinese Journal of Tissue Engineering Research

基　　金：口腔疾病防治全国重点实验室开放课题项目(SKLOD2024OF04),项目负责人:刘旭倩;泸州市科知局科技创新人才计划项目(2023RCX171),项目负责人:刘旭倩;四川省医学科研课题计划项目(S23043),项目负责人:刘旭倩;彭州市-西南医科大学联合项目(2023PZXNYD12),项目负责人:刘旭倩。

摘　　要：背景:前期研究中三维细胞重建组织工程化口腔黏膜等效物结构类似于正常口腔黏膜,即存在类上皮样结构、类固有层样结构、类血管腔样结构,并已初步实现了等效物的血管化建立,但其血管化特征尚不十分明确。目的:采用血管内皮细胞特异性标志物表达谱关联激光捕获显微切割系统靶向获取血管化口腔黏膜等效物的血管样结构,评价其成血管能力,揭示其血管化特征。方法:分别从人牙龈上皮组织和固有层组织原代培养人牙龈上皮细胞、人牙龈成纤维细胞、人牙龈间充质干细胞,人牙龈间充质干细胞经单克隆扩增培养后诱导分化形成血管内皮样细胞。将人牙龈上皮细胞、人牙龈成纤维细胞、血管内皮样细胞分层负载于脱细胞血管基质-0.25%类人Ⅰ型胶原支架上,构建血管化口腔黏膜等效物。将血管化口腔黏膜等效物(实验组)与脱细胞血管基质-0.25%类人Ⅰ型胶原支架(对照组)分别植入裸鼠背部皮下,14 d后两组切口表面涂布生物胶,实验组生物胶表面接种人牙龈上皮细胞,对照组不接种细胞,继续饲养14 d后取材,利用形态学观察口腔黏膜等效物分层结构;采用较为全面的血管内皮细胞特异性标志物表达谱对口腔黏膜等效物中的新生血管样结构进行免疫组化、免疫荧光标记,进行血管化特征分析;采用激光捕获显微切割系统靶向捕获免疫组化特异性标记的口腔黏膜等效物中新生血管样结构,靶向分析其血管化特征。结果与结论:(1)形态学观察显示口腔黏膜等效物细胞层次清晰,结构类似于正常口腔黏膜,即存在类上皮样结构、类固有层样结构、类血管腔样结构,类血管腔样结构内存在散在红细胞;(2)口腔黏膜等效物组中EdU Apollo示踪种子细胞结果显示:EdU Apollo 488标记的人牙龈上皮细胞呈绿色荧光表达;DAPI标记的人牙龈成纤维细胞呈蓝色荧光表达,体内形成类固有层�BACKGROUND:In previous studies,the equivalent structure of three-dimensional cell reconstruction of tissue engineering oral mucosa is similar to normal oral mucosa,including epithelial-like structure,lamina propria-like structure,and vascular lumen-like structure,and has initially achieved the establishment of vascular equivalent,but its vascularization characteristics are not very clear.OBJECTIVE:Vascular-like structures of vascularized oral mucosa equivalent were obtained by targeting vascular endothelial cells specific marker expression profiles correlated with laser capture microdissection system,and their vascularization ability was evaluated to reveal their vascularization characteristics.METHODS:Human gingival epithelial cells were cultured from human gingival epithelium and human gingival fibroblasts,human gingival mesenchymal stem cells were cultured from human gingival lamina propria.Human gingival mesenchymal stem cells were induced to differentiate into vascular endothelial-like cells after monoclonal expansion culture.Human gingival epithelial cells,human gingival fibroblasts,and vascular endothelial-like cells were loaded with acellular vascular matrix-0.25%human-like collagen type I scaffold to construct the vascularized oral mucosa equivalent.The layered structure of oral mucosa equivalent(experimental group)and the acellular vascular matrix-0.25%human-like collagen type I scaffold(control group)were implanted subcutaneously into the back of nude mice,respectively.14 days later,the incision surface of the two groups was coated with biogel.The biogel surface of the experimental group was inoculated with human gingival epithelial cells,while the control group was not inoculated with cells.The samples were collected after 14 days of feeding.The layered structure of oral mucosa equivalent was observed by morphology.The neovascular-like structures in oral mucosa equivalents were labeled by immunohistochemistry and immunofluorescence with a more comprehensive expression profile of vascular endothelial c

关 键 词：脱细胞血管基质 血管内皮样细胞 三维细胞重建 血管化 口腔黏膜等效物 激光捕获显微切割系统 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学] R781.9