人羊膜间充质细胞诱导成骨及与胶原-明胶海绵载体复合的实验研究  

Experimental study on the combination of human amniotic mesenchymal cells induced osteogenesis with collagen-gelatin sponge carrier

作　　者：王芳 宋庆高 郭佳男 陈尚 何苇 邹亚莉 Wang Fang;Song Qinggao;Guo Jianan;Chen Shang;He Wei;Zou Yali(Department of Oral and Maxillofacial Surgery,Hospital of Stomatology,Zunyi Medical University,Zunyi 563000,Guizhou,China)

机构地区：[1]遵义医科大学附属口腔医院口腔颌面外科,贵州遵义563000

出　　处：《贵州医药》2020年第9期1355-1357,F0003,共4页Guizhou Medical Journal

基　　金：贵州省科学技术基金[黔科合J字LKZ(2013)29号]。

摘　　要：目的通过体外诱导的方法促使人羊膜间充质细胞(human amniotic mesenchymal cells,hAMCs)成骨分化,并借用胶原—明胶海绵为载体进行复合培养,观察成骨分化后的hAMCs与胶原—明胶海绵载体的复合情况,旨在寻找修复牙槽突裂的种子细胞。方法结合使用机械法和酶分离法从人羊膜组织中分离hAMCs,将其培养至第三代后利用流式细胞术分选和免疫组织化学的方法进行表型鉴定;加入成骨诱导剂诱导第三代hAMCs,采用茜素红染色检测钙盐沉积的方法判定其成骨分化的程度;将第三代hAMCs消化后接种于胶原—明胶海绵载体上复合培养,观察细胞与胶原—明胶海绵载体复合后的生长情况。结果流式细胞术分选结果显示第三代hAMCs高表达CD29、CD166、CD44、CD73,但不表达CD34、CD45;免疫组织化学结果显示hAMCs波形蛋白染色阳性;茜素红染色结果显示经成骨诱导21 d后可在hAMCs中检测到染色阳性的钙结节;倒置相差显微镜观察显示hAMCs与胶原—明胶海绵在载体复合培养72 h后能形成良好的粘附生长。结论本实验成功从人羊膜组织中分离hAMCs,且培养至第三代的hAMCs符合实验标准,同时成功构建了hAMCs诱导成骨分化模型。此外,胶原—明胶海绵可作为与hAMCs进行复合培养的良好载体,为后续进行动物体内实验提供前期体外实验基础。Objective Human amniotic mesenchymal cells(hAMCs)were induced by in vitro induction to induce osteogenic differentiation,and collagen-gelatin sponge was used as carrier for compound culture to observe the combination of hAMCs and collagen-gelatin sponge carrier after osteogenic differentiation,so as to find seed cells to repair alveolar cleft.Methods HAMCs was isolated from human amniotic tissue by mechanical method and enzyme separation,and its phenotype was identified by flow cytometry and immunohistochemistry after its culture to the third generation.The third generation hAMCs was induced by adding osteogenic inducer,and the degree of osteogenic differentiation was determined by alizarin red staining to detect calcium salt deposition.The third generation of hAMCs was digested and inoculated on the collagen-gelatin sponge carrier for composite culture,and the growth of the cells after the combination with the collagen-gelatin sponge carrier was observed.Results Flow cytometry results showed that the third generation of hAMCs showed high expression of CD29,CD166,CD44 and CD73,but no expression of CD34 and CD45.Immunohistochemistry results showed that hAMCs vimentin staining was positive.Alizarin red staining showed that positive calcium nodules could be detected in hAMCs after 21 days of osteogenic induction.The inverted phase contrast microscope showed that hAMCs and collagen-gelatin sponge could form good adhesion growth after 72 hours of carrier compound culture.Conclusion hAMCs was successfully isolated from human amniotic tissue,and the hAMCs cultured to the third generation met the experimental standards.hAMCs induced osteogenic differentiation was successfully constructed the model of.Collagen-gelatin sponge can be used as a good carrier for compound culture with hAMCs,providing a basis for in vitro experiments at the early stage for subsequent animal experiments.

关 键 词：牙槽突裂 人羊膜间充质细胞 成骨分化 明胶海绵 

分 类 号：R34[医药卫生—基础医学] R782